JP7426973B2 - Hepatitis B virus (HBV) iRNA composition and method of use thereof - Google Patents

Description

Related Applications This application is filed in U.S. Provisional Patent Application No. 62/077,799, filed on November 10, 2014, and U.S. Provisional Patent Application No. 62/137,464, filed on March 24, 2015. claim the benefit of priority over the specification. The entire contents of each of the aforementioned patent applications are hereby incorporated by reference.

This application also claims priority to U.S. Provisional Patent Application No. 62/077,672, filed on November 10, 2014, the entire contents of which are hereby incorporated by reference. Used in the book.

This application relates to PCT/US2015/XXXXX entitled "Hepatitis D Viris (HDV) iRNA Compositions and Methods of Use Thereof" filed on November 10, 2015, and all contents thereof are as follows: Incorporated herein by reference.

SEQUENCE LISTING This application contains a sequence listing submitted electronically in ASCII format, which is hereby incorporated by reference in its entirety. Said ASCII copy created on November 5, 2015 has the name 121301-02320_SL. txt, and the size is 385,791 bytes.

More than 400 million people worldwide are chronically infected with HBV and are therefore at high risk of developing serious liver diseases such as chronic hepatitis, cirrhosis, liver failure and hepatocellular carcinoma (HCC), with an estimated 600,000 deaths have been caused.

The natural evolution of chronic HBV infection involves four sequential stages: (1) an initial "immune tolerance" stage, with high levels of viral replication and minimal hepatitis; (2) an immune response stage, with significant Liver inflammation and elevated serum aminotransferases; some patients progress to the (3) "non-replicating" stage, seroconversion to anti-HBe; undetectable or low-level viremia (<2000 IU/ml by PCR-based assay) ); resolution of liver inflammation; and (4) HBeAg-negative chronic hepatitis B (due to the emergence of specific viral mutations that prevent HBeAg production but not viral replication). This form of chronic hepatitis B (CHB) is characterized by fluctuations in serum HBV DNA and serum aminotransferase (ALT and AST) levels, and progressive liver disease. It is important to note that CHB can exist either as HBeAg positive CHB or as HBeAg negative CHB. Longitudinal studies of CHB patients have shown that the 5-year cumulative incidence of developing cirrhosis ranges from 8-20%. The 5-year cumulative incidence of hepatic decompensation is approximately 20%. The incidence of HCC is increasing worldwide and is currently the fifth most common cancer. The annual incidence of HBV-related HCC is high, ranging from 2-5% in patients with cirrhosis.

The main goal of HBV treatment is to permanently suppress HBV replication and ameliorate liver disease. Clinically important short-term goals are to achieve HBeAg seroconversion, normalization of serum ALT and AST, resolution of hepatitis, and prevent hepatic decompensation. The ultimate goal of treatment is to achieve a sustained response to prevent the development of cirrhosis, liver cancer, and prolong survival. HBV infection cannot be completely eradicated because a specific form of viral covalently closed circular DNA (ccc HBV DNA) persists in the nucleus of infected hepatocytes. However, treatment-induced serum HBsAg clearance is a marker of termination of chronic HBV infection and is associated with the best long-term outcome.

Current standard methods of HBV treatment involve suppression of virus production by interferon or thymosin al-based immunotherapy and inhibition of HBV polymerase. HBV polymerase inhibitors are effective in reducing virus production, but have little to no effect in rapidly reducing HBsAg (as with tenofovir disoproxil fumarate), or in a limited number of patients. HBsAg can be reduced slowly with long-term treatment. Interferon-based immunotherapy can achieve both reduction in virus production and initial blood removal of HBsAg, but only in a small proportion of treated subjects. The generally accepted role of HBsAg in the blood is to sequester anti-HBsAg antibodies and allow infectious virus particles to evade immune detection, which is probably why HBV infection remains chronic. It seems to be one. In addition, HBsAg, HBeAg and HBcAg all have immunoinhibitory properties, and the persistence of these viral proteins in a patient's blood after administering any of the currently available HBV treatments means that the patient may not be aware of their HBV infection. This is likely to have a significant impact in that it prevents achieving immunological control of the disease.

Although all three major HBV proteins (HBsAg, HBeAg and HBcAg) have immunoinhibitory properties, HBsAg accounts for the vast majority of HBV proteins in the circulation of HBV-infected subjects. In addition, while removal of HBeAg (via seroconversion) or reduction of serum viremia is not associated with the development of sustained HBV infection control upon discontinuation of treatment, removal of serum HBsAg from the blood in HBV infection ( seroconversion) is a well-recognized prognostic indicator of antiviral response during treatment that can lead to control of HBV infection upon discontinuation of treatment (although this occurs in a small proportion of patients receiving immunotherapy). ). Therefore, although reduction of all three major HBV proteins (HBsAg, HBeAg and HBcAg) may result in optimal removal of the inhibitory effects, HBsAg Removal alone appears to be sufficient by itself.

Therefore, while there are currently no therapeutic regimens that can restore HBV immunological control in the majority of patients, HBV infections that can inhibit viral replication and restore immunological control in the majority of patients. Effective treatments are needed. Therefore, there is a need in the art for alternative and combination therapies for subjects infected with HBV and/or having HBV-related diseases.

The present invention provides iRNA compositions that produce RNA-induced silencing complex (RISC)-mediated cleavage of RNA transcripts of hepatitis B virus (HBV) genes. The HBV gene may be located within a cell, eg, a cell within the body of a subject, such as a human.

The present invention also relates to disorders that may benefit from inhibiting or reducing the expression of HBV genes, such as HBV infection and/or HBV-related diseases, such as chronic hepatitis B infection (CHB), cirrhosis, liver failure, and hepatocellular carcinoma (HCC) using an iRNA composition that produces RNA-induced silencing complex (RISC)-mediated cleavage of RNA transcripts of HBV genes to inhibit expression of HBV genes. Also provided are methods of treating and therapies.

The RNAi agents of the invention are designed to target regions within the HBV genome that are conserved across all eight serotypes of HBV. In addition, the RNAi agents of the present invention inhibit all stages of the HBV life cycle, such as viral replication, assembly, secretion, and secretion of subviral antigens, by inhibiting the expression of two or more HBV genes. is designed to. In particular, since polycistronic overlapping RNAs are generated when the HBV genome is transcribed, the RNAi agents of the present invention that target a single HBV gene will significantly inhibit the expression of most or all HBV transcripts. It turns out. For example, because the HBV genome is transcribed into a single mRNA, the RNAi agents of the invention that target the S gene will inhibit not only S gene expression, but also expression of the "downstream" reverse transcriptase gene. Become. Furthermore, the RNAi agents of the present invention inhibit HBV viral replication by targeting HBV structural genes and the HBV It is designed to be able to produce and eliminate HBV infection. Without intending to be limited by theory, it is believed that combinations or subcombinations of the foregoing properties and specific target sites and/or specific modifications of these RNAi agents may provide efficacy for the RNAi agents of the present invention. , are believed to confer improved stability, safety, efficacy, and persistence.

Accordingly, in one aspect, the invention provides double-stranded RNAi agents for inhibiting hepatitis B virus (HBV) expression in cells. The double-stranded RNAi agent comprises a sense strand and an antisense strand forming a double-stranded region, the sense strand comprising at least 15 consecutive nucleotides that differ from the nucleotide sequence of SEQ ID NO: 1 by no more than 3 nucleotides; and said antisense strand comprises at least 15 contiguous nucleotides that differ from the nucleotide sequence of SEQ ID NO: 2 by no more than 3 nucleotides, substantially all of the nucleotides of said sense strand and substantially all of the nucleotides of said antisense strand. is a modified nucleotide, the sense strand is conjugated to a ligand attached at the 3' end, and the ligand is one or more nucleotides attached via a divalent or trivalent branched linker. It is a GalNAc derivative.

In one embodiment, one or more of the three nucleotide differences in the nucleotide sequence of the antisense strand is a nucleotide mismatch in the antisense strand.

In another embodiment, one or more of the three nucleotide differences in the nucleotide sequence of the antisense strand is a nucleotide mismatch in the sense strand.

In one embodiment, all of the nucleotides of the sense strand and all of the nucleotides of the antisense strand are modified nucleotides.

In one embodiment, the sense strand and antisense strand include any one of the sequences listed in any one of Tables 3, 4, 6, 7, 12, 13, 22, 23, 25, and 26. It includes a region of complementarity comprising at least 15 contiguous nucleotides that differ by no more than 1 nucleotide.

In one embodiment, the at least one modified nucleotide is a deoxy-nucleotide, a 3' terminal deoxy-thymine (dT) nucleotide, a 2'-O-methyl modified nucleotide, a 2'-fluoro modified nucleotide, a 2'-deoxy-modified nucleotide. , fixed nucleotide, non-fixed nucleotide, conformationally restricted nucleotide, constrained ethyl nucleotide, non-basic nucleotide, 2'-amino-modified nucleotide, 2'-O-allyl-modified nucleotide, 2'-C- Alkyl-modified nucleotides, 2'-hydroxyl-modified nucleotides, 2'-methoxyethyl-modified nucleotides, 2'-O-alkyl-modified nucleotides, morpholinonucleotides, phosphoroamidates, nucleotides containing unnatural bases, tetrahydropyran-modified nucleotides , 1,5-anhydrohexitol modified nucleotides, cyclohexenyl modified nucleotides, nucleotides containing a phosphorothioate group, nucleotides containing a methylphosphonate group, nucleotides containing a 5'-phosphate, and nucleotides containing a 5'-phosphate mimetic. selected from the group.

In one embodiment, at least one strand includes a 3' overhang of at least one nucleotide. In another embodiment, at least one strand includes a 3' overhang of at least two nucleotides.

In one embodiment, the double-stranded region is 15-30 nucleotide pairs long. In another embodiment, the double-stranded region is 17-23 nucleotide pairs long. In yet another embodiment, the double-stranded region is 17-25 nucleotide pairs in length. In one embodiment, the double-stranded region is 23-27 nucleotide pairs long. In another embodiment, the double-stranded region is 19-21 nucleotide pairs long. In yet another embodiment, the double-stranded region is 21-23 nucleotide pairs long.

In one embodiment, each strand has 15-30 nucleotides. In another embodiment, each strand has 19-30 nucleotides.

である。 In one embodiment, the ligand is

It is.

に示されるリガンドにコンジュゲートされ、式中、ＸはＯ又はＳである。 In one embodiment, the RNAi agent is shown in the schematic diagram below.

where X is O or S.

In one embodiment, the RNAi agent is selected from the group of RNAi agents listed in any one of Tables 3, 4, 6, 7, 12, 13, 22, 23, 25, and 26.

In one aspect, the invention provides double-stranded RNAi agents for inhibiting hepatitis B virus (HBV) expression in cells. This double-stranded RNAi agent comprises a sense strand and an antisense strand forming a double-stranded region, the sense strand comprising 5'-UCGUGGUGGACUUCUCUCA-3' (SEQ ID NO: 5), and the antisense strand comprising 5'-UCGUGGUGGACUUCUCCA-3' (SEQ ID NO: 5). '-UGAGAGAAGUCCACCACGAUU-3' (SEQ ID NO: 6), substantially all of the nucleotides of the sense strand and substantially all of the nucleotides of the antisense strand are modified nucleotides; and the ligand is one or more GalNAc derivatives attached via a divalent or trivalent branched linker.

The present invention also provides embodiments that are at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical over their entire length to the aforementioned sense and antisense nucleotide sequences. RNAi agents including sense and antisense nucleotide sequences are also provided.

In another aspect, the invention provides double-stranded RNAi agents for inhibiting hepatitis B virus (HBV) expression in cells. This double-stranded RNAi agent includes a sense strand and an antisense strand forming a double-stranded region, the sense strand includes 5'-GUGCACUUCGCUUCACCUCUA-3' (SEQ ID NO: 7), and the antisense strand includes 5'-GUGCACUUCGCUUCACCUCUA-3' (SEQ ID NO: 7). '-UAGAGGUGAAGCGAAGUGCACUU-3' (SEQ ID NO: 8), substantially all of the nucleotides of the sense strand and substantially all of the nucleotides of the antisense strand are modified nucleotides, and the sense strand has a 3' end and the ligand is one or more GalNAc derivatives attached via a divalent or trivalent branched linker. The present invention also provides embodiments that are at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical over their entire length to the aforementioned sense and antisense nucleotide sequences. RNAi agents including sense and antisense nucleotide sequences are also provided.

In another aspect, the invention provides double-stranded RNAi agents for inhibiting hepatitis B virus (HBV) expression in cells. This double-stranded RNAi agent includes a sense strand and an antisense strand forming a double-stranded region, the sense strand includes 5'-CGUGGUGGACUUCUCUCAAUU-3' (SEQ ID NO: 9), and the antisense strand includes 5 '-AAUUGAGAGAAGUCCACCAGCAG-3' (SEQ ID NO: 10), substantially all of the nucleotides of the sense strand and substantially all of the nucleotides of the antisense strand are modified nucleotides, and the sense strand has a 3' end and the ligand is one or more GalNAc derivatives attached via a divalent or trivalent branched linker. The present invention also provides embodiments that are at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical over their entire length to the aforementioned sense and antisense nucleotide sequences. RNAi agents including sense and antisense nucleotide sequences are also provided.

In another aspect, the invention provides double-stranded RNAi agents for inhibiting hepatitis B virus (HBV) expression in cells. This double-stranded RNAi agent includes a sense strand and an antisense strand forming a double-stranded region, the sense strand includes 5'-CGUGGUGGCUUCUCUAAAUU-3' (SEQ ID NO: 37), and the antisense strand includes 5'- AAUUGAGAGAGAAGUCCACCAGCUU-3' (SEQ ID NO: 38),
Substantially all of the nucleotides of the sense strand and substantially all of the nucleotides of the antisense strand are modified nucleotides, the sense strand is conjugated at the 3' end to an attached ligand, and the ligand is divalent. or one or more GalNAc derivatives attached via a trivalent branched linker. The present invention also provides embodiments that are at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical over their entire length to the aforementioned sense and antisense nucleotide sequences. RNAi agents including sense and antisense nucleotide sequences are also provided.

In another aspect, the invention provides double-stranded RNAi agents for inhibiting hepatitis B virus (HBV) expression in cells. This double-stranded RNAi agent includes a sense strand and an antisense strand forming a double-stranded region, the sense strand includes 5'-GGUGGACUUCUCUCAAUUUUA-3' (SEQ ID NO: 11), and the antisense strand includes 5'-GGUGGACUUCUCUCAAUUUUA-3' (SEQ ID NO: 11). '-UAAAAUUGAGAGAGAAGUCCACCAC-3' (SEQ ID NO: 12), substantially all of the nucleotides of the sense strand and substantially all of the nucleotides of the antisense strand are modified nucleotides, and the sense strand has a 3' end and the ligand is one or more GalNAc derivatives attached via a divalent or trivalent branched linker. The present invention also provides embodiments that are at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical over their entire length to the aforementioned sense and antisense nucleotide sequences. RNAi agents including sense and antisense nucleotide sequences are also provided.

In another aspect, the invention provides double-stranded RNAi agents for inhibiting hepatitis B virus (HBV) expression in cells. This double-stranded RNAi agent comprises a sense strand and an antisense strand forming a double-stranded region, the sense strand comprising 5'-GUGUGCACUUCGCUUCACA-3' (SEQ ID NO: 39), and the antisense strand comprising 5'-GUGUGCACUUCGCUUCACA-3' (SEQ ID NO: 39). '-UGUGAAGCGAAGUGCACACUU-3' (SEQ ID NO: 40), substantially all of the nucleotides of the sense strand and substantially all of the nucleotides of the antisense strand are modified nucleotides, and the sense strand has a 3' end and the ligand is one or more GalNAc derivatives attached via a divalent or trivalent branched linker. The present invention also provides embodiments that are at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical over their entire length to the aforementioned sense and antisense nucleotide sequences. RNAi agents including sense and antisense nucleotide sequences are also provided.

In one embodiment, all of the nucleotides of the sense strand and all of the nucleotides of the antisense strand include modified nucleotides.

In certain embodiments, the modified nucleotides are 3' terminal deoxy-thymine (dT) nucleotides, 2'-O-methyl modified nucleotides, 2'-fluoro modified nucleotides, 2'-deoxy-modified nucleotides, fixed nucleotides, non-fixed nucleotides. , conformationally restricted nucleotide, constrained ethyl nucleotide, abasic nucleotide, 2'-amino-modified nucleotide, 2'-O-allyl-modified nucleotide, 2'-C-alkyl-modified nucleotide, 2' -Hydroxyl-modified nucleotides, 2'-methoxyethyl-modified nucleotides, 2'-O-alkyl-modified nucleotides, morpholinonucleotides, phosphoroamidates, nucleotides containing unnatural bases, tetrahydropyran-modified nucleotides, 1,5-anhydro selected from the group consisting of hexitol modified nucleotides, cyclohexenyl modified nucleotides, nucleotides containing phosphorothioate groups, nucleotides containing methylphosphonate groups, nucleotides containing 5'-phosphate, and nucleotides containing 5'-phosphate mimetics.

In one embodiment, the 5'-phosphate mimetic is 5'-vinyl phosphate (5'-VP).

In one embodiment, the sense strand comprises 5'-uscsguGfgUfGfGfacuucucuca-3' (SEQ ID NO: 13) and the antisense strand comprises 5'-usGfsagaGfaAfGfuccaCfcAfcgasusu-3' (SEQ ID NO: 14) [wherein A, C, G and U are ribose A, C, G or U; a, g, c and u are 2'-O-methyl (2'-OMe) A, U, C or G; Af , Cf, Gf or Uf is 2'-fluoro A, G, C or U; and s is a phosphorothioate bond].

In another embodiment, the sense strand comprises 5'-uscsguGfgUfGfGfacuucucuca-3' (SEQ ID NO: 15) and the antisense strand comprises 5'-PusGfsagaGfaAfGfuccaCfcAfcgasusu-3' (SEQ ID NO: 16) [wherein A, C , G, and U are ribose A, C, G, or U; a, g, c, and u are 2'-O-methyl (2'-OMe) A, U, C, or G; Af, Cf, Gf or Uf is 2'-fluoro A, G, C or U; and s is a phosphorothioate bond; and P is 5'-phosphate or a 5' phosphate mimetic].

In one embodiment, the sense strand comprises 5'-gsusgcacUfuCfGfCfuucaccucua-3' (SEQ ID NO: 17) and the antisense strand comprises 5'-usAfsgagGfugaagcgAfaGfugcacsusu-3' (SEQ ID NO: 18) [wherein A, C, G and U are ribose A, C, G or U; a, g, c and u are 2'-O-methyl (2'-OMe) A, U, C or G; Af , Cf, Gf or Uf is 2'-fluoro A, G, C or U; and s is a phosphorothioate bond].

In another embodiment, the sense strand comprises 5'-gsusgcacUfuCfGfCfuucaccucua-3' (SEQ ID NO: 19) and the antisense strand comprises 5'-PusAfsgagGfugaagcgAfaGfugcacsusu-3' (SEQ ID NO: 20) [wherein A, C , G, and U are ribose A, C, G, or U; a, g, c, and u are 2'-O-methyl (2'-OMe) A, U, C, or G; Af, Cf, Gf or Uf is 2'-fluoro A, G, C or U; and s is a phosphorothioate bond; and P is 5'-phosphate or a 5' phosphate mimetic].

In one embodiment, the sense strand comprises 5'-csgsugguGfgAfCfUfucucUfCfaauu-3' (SEQ ID NO: 21) and the antisense strand comprises 5'-asAfsuugAfgAfgAfaguCfcAfccagcsasg-3' (SEQ ID NO: 22) [wherein A, C, G and U are ribose A, C, G or U; a, g, c and u are 2'-O-methyl (2'-OMe) A, U, C or G; Af , Cf, Gf or Uf is 2'-fluoro A, G, C or U; and s is a phosphorothioate bond].

In another embodiment, the sense strand comprises 5'-csgsugguGfgAfCfUfucucUfCfaauu-3' (SEQ ID NO: 23) and the antisense strand comprises 5'-PasAfsuugAfgAfgAfaguCfcAfccagcsasg-3' (SEQ ID NO: 24) [wherein A, C , G, and U are ribose A, C, G, or U; a, g, c, and u are 2'-O-methyl (2'-OMe) A, U, C, or G; Af, Cf, Gf or Uf is 2'-fluoro A, G, C or U; and s is a phosphorothioate bond; and P is 5'-phosphate or a 5' phosphate mimetic].

In another embodiment, the sense strand comprises 5'-csgsuggudGgucdTucucuaaauu-3' (SEQ ID NO: 35) and the antisense strand comprises 5'-asdAsuugagagdAagudCcaccagcsusu-3' (SEQ ID NO: 36) [wherein A,C , G, and U are ribose A, C, G, or U; a, g, c, and u are 2'-O-methyl (2'-OMe) A, U, C, or G; dA, dC, dG, and dT are deoxyribose A, C, G, and T; and s is a phosphorothioate bond].

In one embodiment, the sense strand comprises 5'-gsgsuggaCfuUfCfUfcucaAfUfuuua-3' (SEQ ID NO: 25) and the antisense strand comprises 5'-usAfsaaaUfuGfAfgagaAfgUfccaccsasc-3' (SEQ ID NO: 26) [wherein A, C, G and U are ribose A, C, G or U; a, g, c and u are 2'-O-methyl (2'-OMe) A, U, C or G; Af , Cf, Gf or Uf is 2'-fluoro A, G, C or U; and s is a phosphorothioate bond].

In another embodiment, the sense strand comprises 5'-gsgsuggaCfuUfCfUfcucaAfUfuuua-3' (SEQ ID NO: 27) and the antisense strand comprises 5'-PusAfsaaaUfuGfAfgagaAfgUfccaccsasc-3' (SEQ ID NO: 28) [wherein A, C , G, and U are ribose A, C, G, or U; a, g, c, and u are 2'-O-methyl (2'-OMe) A, U, C, or G; Af, Cf, Gf or Uf is 2'-fluoro A, G, C or U; and s is a phosphorothioate bond; and P is 5'-phosphate or a 5' phosphate mimetic].

In another embodiment, the sense strand comprises 5'-gsusguGfcAfCfUfucgcuucaca-3' (SEQ ID NO: 41) and the antisense strand comprises 5'-usGfsugaAfgCfGfaaguGfcAfcacsusu-3' (SEQ ID NO: 42) [wherein A, C , G, and U are ribose A, C, G, or U; a, g, c, and u are 2'-O-methyl (2'-OMe) A, U, C, or G; Af, Cf, Gf or Uf is 2'-fluoro A, G, C or U; and s is a phosphorothioate bond].

である。 In one embodiment, the ligand is

It is.

に示されるリガンドにコンジュゲートされ、式中、ＸはＯ又はＳである。 In one embodiment, the RNAi agent is shown in the schematic diagram below.

where X is O or S.

In one embodiment, P is a 5'-phosphate mimetic. In one embodiment, the 5'-phosphate mimetic is 5'-vinyl phosphate (5'-VP).

In another aspect, the invention provides a composition comprising two or more double-stranded RNAi agents for inhibiting hepatitis B virus (HBV) expression in a cell, each double-stranded RNAi agent comprising: independently comprising a sense strand and an antisense strand forming a double-stranded region, each of said sense strands independently comprising at least 15 contiguous nucleotides that differ from the nucleotide sequence of SEQ ID NO: 1 by no more than 3 nucleotides; and each of said antisense strands independently comprises at least 15 contiguous nucleotides that differ by no more than 3 nucleotides from the nucleotide sequence of SEQ ID NO: 2, and substantially all of the nucleotides of each of said sense strands and said Substantially all of the nucleotides of each of the antisense strands are independently modified nucleotides, each of the sense strands is independently conjugated to a ligand attached at the 3' end, and the ligand is , one or more GalNAc derivatives attached via a divalent or trivalent branched linker.

In one embodiment, one or more of the three nucleotide differences in the nucleotide sequence of the antisense strand is a nucleotide mismatch in the antisense strand. In another embodiment, one or more of the three nucleotide differences in the nucleotide sequence of the antisense strand is a nucleotide mismatch in the sense strand.

In one embodiment, all of the nucleotides of the sense strand and all of the nucleotides of the antisense strand are modified nucleotides.

In one embodiment, the sense strand and the antisense strand are any one of the sequences listed in any one of Tables 3, 4, 6, 7, 12, 13, 22, 23, 25, and 26. Includes a region of complementarity that includes at least 15 contiguous nucleotides that differ by no more than 3 nucleotides.

In one embodiment, at least one of the modified nucleotides is a deoxynucleotide, a 3' terminal deoxythymine (dT) nucleotide, a 2'-O-methyl modified nucleotide, a 2'-fluoro modified nucleotide, a 2'-deoxy modified nucleotide, Fixed nucleotides, non-fixed nucleotides, conformationally restricted nucleotides, constrained ethyl nucleotides, non-basic nucleotides, 2'-amino modified nucleotides, 2'-O-allyl modified nucleotides, 2'-C-alkyl modified nucleotides , 2'-hydroxyl modified nucleotide, 2'-methoxyethyl modified nucleotide, 2'-O-alkyl modified nucleotide, morpholino nucleotide, phosphoramidate, nucleotide containing unnatural base, tetrahydropyran modified nucleotide, 1, From the group consisting of 5-anhydrohexitol modified nucleotides, cyclohexenyl modified nucleotides, nucleotides containing a phosphorothioate group, nucleotides containing a methylphosphonate group, nucleotides containing a 5'-phosphate, and nucleotides containing a 5'-phosphate mimetic. selected.

In another aspect, the invention provides a composition for inhibiting hepatitis B virus (HBV) expression in a cell, the composition comprising: (a) a first sense strand forming a double-stranded region; A first double-stranded RNAi agent comprising a first antisense strand, wherein substantially all of the nucleotides of the first sense strand and substantially all of the nucleotides of the first antisense strand are modified nucleotides. wherein the first sense strand is conjugated to a ligand attached at the 3' end, and the ligand is conjugated to one or more strands attached via a divalent or trivalent branched linker. a first double-stranded RNAi agent that is a GalNAc derivative; and (b) a second double-stranded RNAi agent comprising a second sense strand and a second antisense strand forming a double-stranded region, , substantially all of the nucleotides of the second sense strand and substantially all of the nucleotides of the second antisense strand are modified nucleotides, and the second sense strand is attached at the 3' end. a second double-stranded RNAi agent conjugated to the ligand, and the ligand is one or more GalNAc derivatives attached via a divalent or trivalent branched linker; and the second sense strand each independently,
5'-UCGUGGUGGACUUCUCUCA-3' (SEQ ID NO: 5),
5'-GUGCACUUCGCUUCACCUCUA-3' (SEQ ID NO: 7),
5'-CGUGGUGGACUUCUCUCAAUU-3' (SEQ ID NO: 9),
5'-CGUGGUGGCUUCUCUAAAUU-3' (SEQ ID NO: 37),
5'-GGUGGACUUCUCUCAAUUUUA-3' (SEQ ID NO: 11), and 5'-GUGUGCACUUCGCUUCACA-3' (SEQ ID NO: 39) , 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical nucleotide sequences), and the first and second antisense strands each independently:
5'-UGAGAGAAGUCCACCACGAUU-3' (SEQ ID NO: 6),
5'-UAGAGGUGAAGCGAAGUGCACUU-3' (SEQ ID NO: 8),
5'-AAUUGAGAGAAGUCCACCAGCAG-3' (SEQ ID NO: 10),
5'-AAUUGAGAGAAGUCCACCAGCUU-3' (SEQ ID NO: 38),
5'-UAAAAUUGAGAGAAGUCCACCAC-3' (SEQ ID NO: 12), and 5'-UGUGAAGCGAAGUGCACACUU-3' (SEQ ID NO: 40) , 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical nucleotide sequences).

In one embodiment, all of the nucleotides of the first and second sense strands and/or the first and second antisense strands contain modifications.

In one embodiment, at least one of the modified nucleotides is a deoxynucleotide, a 3' terminal deoxythymine (dT) nucleotide, a 2'-O-methyl modified nucleotide, a 2'-fluoro modified nucleotide, a 2'-deoxy modified nucleotide, Fixed nucleotides, non-fixed nucleotides, conformationally restricted nucleotides, constrained ethyl nucleotides, non-basic nucleotides, 2'-amino modified nucleotides, 2'-O-allyl modified nucleotides, 2'-C-alkyl modified nucleotides , 2'-hydroxyl modified nucleotide, 2'-methoxyethyl modified nucleotide, 2'-O-alkyl modified nucleotide, morpholino nucleotide, phosphoramidate, nucleotide containing unnatural base, tetrahydropyran modified nucleotide, 1, From the group consisting of 5-anhydrohexitol modified nucleotides, cyclohexenyl modified nucleotides, nucleotides containing a phosphorothioate group, nucleotides containing a methylphosphonate group, nucleotides containing a 5'-phosphate, and nucleotides containing a 5'-phosphate mimetic. selected.

In one embodiment, the first and second RNAi agents are
5'-uscsguGfgUfGfGfacuucucuca-3' (SEQ ID NO: 13)
5'-usGfsagaGfaAfGfuccaCfcAfcgasusu-3' (SEQ ID NO: 14);
5'-uscsguGfgUfGfGfacuucucuca-3' (SEQ ID NO: 15)
5'-PusGfsagaGfaAfGfuccaCfcAfcgasusu-3' (SEQ ID NO: 16);
5'-gsusgcacUfuCfGfCfuucaccucua-3' (SEQ ID NO: 17)
5'-usAfsgagGfugaagcgAfaGfugcacsusu-3' (SEQ ID NO: 18);
5'-gsusgcacUfuCfGfCfuucaccucua-3' (SEQ ID NO: 19)
5'-PusAfsgagGfugaagcgAfaGfugcacsusu-3' (SEQ ID NO: 20);
5'-csgsugguGfgAfCfUfucucUfCfauu-3' (SEQ ID NO: 21)
5'-asAfsuugAfgAfgAfaguCfcAfccagcsasg-3' (SEQ ID NO: 22);
5'-csgsugguGfgAfCfUfucucUfCfauu-3' (SEQ ID NO: 23)
5'-PasAfsuugAfgAfgAfaguCfcAfccagcsasg-3' (SEQ ID NO: 24);
5'-csgsuggudGgucdTucucuaaauu-3' (SEQ ID NO: 35)
5'-asdAsuugagagdAagudCcaccagcsusu-3' (SEQ ID NO: 36);
5'-gsgsuggaCfuUfCfUfcucaAfUfuuua-3' (SEQ ID NO: 25)
5'-usAfsaaaUfuGfAfgagaAfgUfccaccsasc-3' (SEQ ID NO: 26);
5'-gsgsuggaCfuUfCfUfcucaAfUfuuua-3' (SEQ ID NO: 27)
5'-PusAfsaaaUfuGfAfgagaAfgUfccaccsasc-3' (SEQ ID NO: 28); and 5'-gsusguGfcAfCfUfucgcuucaca-3' (SEQ ID NO: 41)
5'-usGfsugaAfgCfGfaaguGfcAfcacsusu-3' (SEQ ID NO: 42) [wherein A, C, G, and U are ribose A, C, G, or U; a, g, c, and u are 2'-O -Methyl (2'-OMe) A, U, C, or G; Af, Cf, Gf or Uf is 2'-fluoro A, G, C, or U; dA, dC, dG, and dT are deoxyribose A, C, G, and T; s is a phosphorothioate bond; and P is a 5'-phosphate or a 5' phosphate mimetic.

In one embodiment, the first and second RNAi agents are
5'-uscsguGfgUfGfGfacuucucuca-3' (SEQ ID NO: 15)
5'-PusGfsagaGfaAfGfuccaCfcAfcgasusu-3' (SEQ ID NO: 16);
5'-csgsugguGfgAfCfUfucucUfCfauu-3' (SEQ ID NO: 21)
5'-asAfsuugAfgAfgAfaguCfcAfccagcsasg-3' (SEQ ID NO: 22) [wherein A, C, G, and U are ribose A, C, G, or U; a, g, c, and u are 2'-O - methyl (2'-OMe) is A, U, C or G; Af, Cf, Gf or Uf is 2'-fluoro A, G, C or U; s is a phosphorothioate bond; and P is a 5'-phosphate or a 5' phosphate mimetic].

In another embodiment, the first and second RNAi agents are
5'-gsgsuggaCfuUfCfUfcucaAfUfuuua-3' (SEQ ID NO: 25)
5'-usAfsaaaUfuGfAfgagaAfgUfccaccsasc-3' (SEQ ID NO: 26); and 5'-gsusguGfcAfCfUfucgcuucaca-3' (SEQ ID NO: 41)
5'-usGfsugaAfgCfGfaaguGfcAfcacsusu-3' (SEQ ID NO: 42) [wherein A, C, G, and U are ribose A, C, G, or U; a, g, c, and u are 2'-O - methyl (2'-OMe) is A, U, C or G; Af, Cf, Gf or Uf is 2'-fluoro A, G, C or U; s is a phosphorothioate bond; and P is a 5'-phosphate or a 5' phosphate mimetic].

In one aspect, the invention provides a double-stranded RNAi agent comprising an RNAi agent listed in any one of Tables 3, 4, 6, 7, 12, 13, 22, 23, 25, and 26. .

The invention also provides vectors and cells containing the double-stranded RNAi agents of the invention.

In another aspect, the invention provides a pharmaceutical composition comprising a double-stranded RNAi agent of the invention, or a composition of the invention, or a vector of the invention.

In one embodiment, the double-stranded RNAi agent is administered in an unbuffered solution. In one embodiment, the unbuffered solution is saline or water.

In another embodiment, the double-stranded RNAi agent is administered with a buffer. In one embodiment, the buffer comprises acetate, citrate, prolamine, carbonate, or phosphate or any combination thereof. In another embodiment, the buffer is phosphate buffered saline (PBS).

In one aspect, the invention provides a method of inhibiting hepatitis B virus (HBV) gene expression in a cell. This method comprises the steps of: contacting a cell with a double-stranded RNAi agent of the present invention, a composition of the present invention, a vector of the present invention, or a pharmaceutical composition of the present invention; maintaining the mRNA transcript for a sufficient period of time to obtain degradation, thereby inhibiting HBV gene expression in the cell.

In one embodiment, the HBV gene is selected from the group consisting of C, X, P, S, and combinations thereof.

In one aspect, the invention provides a method of inhibiting hepatitis B virus (HBV) replication in a cell. This method comprises the steps of: contacting a cell with a double-stranded RNAi agent of the present invention, a composition of the present invention, a vector of the present invention, or a pharmaceutical composition of the present invention; and maintaining the mRNA transcript for a sufficient period of time to obtain degradation of the mRNA transcript, thereby inhibiting HBV replication in the cell.

In one embodiment, the cell is within the subject's body. In one embodiment, the subject is a human.

In one embodiment, the subject is suffering from an HBV-related disease.

In one embodiment, HBV gene expression is at least about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 98% or about 100%. inhibited.

In one embodiment, HBV replication in the cell is at least about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 98%, or about 100% inhibited.

In one aspect, the invention provides a method of reducing hepatitis B virus (HBV) DNA levels in a subject infected with HBV. The method comprises administering to a subject a therapeutically effective amount of a double-stranded RNAi agent of the invention, or a composition of the invention, or a vector of the invention, or a pharmaceutical composition of the invention, thereby reducing the HBV ccc DNA of the subject. including the step of reducing the level.

In another aspect, the invention provides a method of reducing hepatitis B virus (HBV) antigen levels in a subject infected with HBV. The method involves administering to a subject a therapeutically effective amount of a double-stranded RNAi agent of the invention, or a composition of the invention, or a vector of the invention, or a pharmaceutical composition of the invention, thereby increasing the HBV antigen level in the subject. including the step of reducing.

In one embodiment, the HBV antigen is HBsAg. In another embodiment, the HBV antigen is HBeAg.

In another aspect, the invention provides a method of reducing hepatitis B virus (HBV) viral load in a subject infected with HBV. The method comprises administering to a subject a therapeutically effective amount of a double-stranded RNAi agent of the invention, or a composition of the invention, or a vector of the invention, or a pharmaceutical composition of the invention, thereby reducing the HBV viral load of the subject. including the step of reducing.

In yet another aspect, the invention provides a method of reducing alanine aminotransferase (ALT) levels in a subject infected with HBV. This method involves administering to a subject a therapeutically effective amount of a double-stranded RNAi agent of the present invention, or a composition of the present invention, or a vector of the present invention, or a pharmaceutical composition of the present invention, thereby reducing the ALT level of the subject. including the step of reducing.

In another aspect, the invention provides a method of reducing aspartate aminotransferase (AST) levels in a subject infected with HBV. This method involves administering to a subject a therapeutically effective amount of a double-stranded RNAi agent of the present invention, or a composition of the present invention, or a vector of the present invention, or a pharmaceutical composition of the present invention, thereby reducing the AST level in the subject. including the step of reducing.

In another aspect, the invention provides a method of increasing anti-hepatitis B virus (HBV) antibody levels in a subject infected with HBV. This method involves administering to a subject a therapeutically effective amount of a double-stranded RNAi agent of the present invention, or a composition of the present invention, or a vector of the present invention, or a pharmaceutical composition of the present invention, thereby increasing the anti-HBV antibody of the subject. including the step of increasing the level.

In one aspect, the invention provides a method of treating a subject with a hepatitis B virus (HBV) infection. This method comprises administering to a subject a therapeutically effective amount of a double-stranded RNAi agent of the present invention, or a composition of the present invention, or a vector of the present invention, or a pharmaceutical composition of the present invention, thereby treating said subject. Including process.

In another aspect, the invention provides a method of treating a subject with a hepatitis B virus (HBV)-related disorder. This method comprises administering to a subject a therapeutically effective amount of a double-stranded RNAi agent of the present invention, or a composition of the present invention, or a vector of the present invention, or a pharmaceutical composition of the present invention, thereby treating said subject. Including process.

In one embodiment, the HBV-related disorder is the group consisting of hepatitis D virus infection, delta hepatitis, acute hepatitis B; acute fulminant hepatitis B; chronic hepatitis B; liver fibrosis; end-stage liver disease; hepatocellular carcinoma. selected from.

In one embodiment, the HBV-related disorder is chronic hepatitis and the subject is HBeAg positive. In another embodiment, the HBV-related disorder is chronic hepatitis and the subject is HBeAg negative.

In one aspect, the invention provides a method of treating a subject with a hepatitis B virus (HBV) infection. The method comprises a therapeutically effective amount of a double-stranded RNAi agent [the double-stranded RNAi agent comprises a sense strand and an antisense strand forming a double-stranded region;
The sense strand is 5'-UCGUGGUGGACUUCUCUCA-3' (SEQ ID NO: 5) (or the aforementioned nucleotide sequence and at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% over its entire length). . 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical nucleotide sequence), substantially all of the nucleotides of the sense strand and of the nucleotides of the antisense strand. Substantially all are modified nucleotides, the sense strand is conjugated to a ligand attached at the 3' end, and the ligand is a nucleotide attached via a divalent or trivalent branched linker. one or more GalNAc derivatives] to the subject, thereby treating the subject.

In another aspect, the invention provides a method of treating a subject with a hepatitis B virus (HBV)-related disorder. The method comprises administering a therapeutically effective amount of a double-stranded RNAi agent [the double-stranded RNAi agent comprises a sense strand and an antisense strand forming a double-stranded region, the sense strand being 5'-UCGUGGUGGACUUCUCUCA-3' ( SEQ ID NO: 5) (or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical over its entire length to the aforementioned nucleotide sequence) and the antisense strand comprises 5'-UGAGAGAAGUCCACCACGAUU-3' (SEQ ID NO: 6) (or the aforementioned nucleotide sequence and at least 90%, 91%, 92%, 93%, 94%, 95% over its entire length, 96%, 97%, 98%, or 99% identical nucleotide sequence), substantially all of the nucleotides of the sense strand and substantially all of the nucleotides of the antisense strand are modified nucleotides; the chain is conjugated to a ligand attached at the 3' end, and the ligand is one or more GalNAc derivatives attached via a divalent or trivalent branched linker. administering and thereby treating the subject.

In one aspect, the invention provides a method of treating a subject with a hepatitis B virus (HBV) infection. The method comprises a therapeutically effective amount of a double-stranded RNAi agent [the double-stranded RNAi agent comprises a sense strand and an antisense strand forming a double-stranded region;
The sense strand is 5'-GUGCACUUCGCUUCACCUCUA-3' (SEQ ID NO: 7) (or the aforementioned nucleotide sequence and at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% over its entire length). . 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical nucleotide sequence), substantially all of the nucleotides of the sense strand and of the nucleotides of the antisense strand. Substantially all are modified nucleotides, the sense strand is conjugated to a ligand attached at the 3' end, and the ligand is a nucleotide attached via a divalent or trivalent branched linker. one or more GalNAc derivatives] to the subject, thereby treating the subject.

In another aspect, the invention provides a method of treating a subject with a hepatitis B virus (HBV)-related disorder. The method comprises a therapeutically effective amount of a double-stranded RNAi agent [the double-stranded RNAi agent comprises a sense strand and an antisense strand forming a double-stranded region;
The sense strand is 5'-GUGCACUUCGCUUCACCUCUA-3' (SEQ ID NO: 7) (or the aforementioned nucleotide sequence and at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% over its entire length). . 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical nucleotide sequence), substantially all of the nucleotides of the sense strand and of the nucleotides of the antisense strand. Substantially all are modified nucleotides, the sense strand is conjugated to a ligand attached at the 3' end, and the ligand is a nucleotide attached via a divalent or trivalent branched linker. one or more GalNAc derivatives] to the subject, thereby treating the subject.

In one aspect, the invention provides a method of treating a subject with a hepatitis B virus (HBV) infection. The method comprises a therapeutically effective amount of a double-stranded RNAi agent [the double-stranded RNAi agent comprises a sense strand and an antisense strand forming a double-stranded region;
The sense strand is 5'-CGUGGUGGACUUCUCUCAAUU-3' (SEQ ID NO: 9) (or the aforementioned nucleotide sequence and at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% over its entire length). , 98%, or 99% identical nucleotide sequence), and said antisense strand comprises 5'-AAUUGAGAGAGAAGUCCACCAGCAG-3' (SEQ ID NO: 10) (or at least 90%, 91%, over its entire length with said nucleotide sequence); 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical nucleotide sequence), substantially all of the nucleotides of the sense strand and of the nucleotides of the antisense strand. Substantially all are modified nucleotides, the sense strand is conjugated to a ligand attached at the 3' end, and the ligand is a nucleotide attached via a divalent or trivalent branched linker. one or more GalNAc derivatives] to the subject, thereby treating the subject.

In another aspect, the invention provides a method of treating a subject with a hepatitis B virus (HBV)-related disorder. The method comprises a therapeutically effective amount of a double-stranded RNAi agent [the double-stranded RNAi agent comprises a sense strand and an antisense strand forming a double-stranded region;
The sense strand is 5'-CGUGGUGGACUUCUCUCAAUU-3' (SEQ ID NO: 9) (or the aforementioned nucleotide sequence and at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% over its entire length). . 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical nucleotide sequence), substantially all of the nucleotides of the sense strand and of the nucleotides of the antisense strand. Substantially all are modified nucleotides, the sense strand is conjugated to a ligand attached at the 3' end, and the ligand is a nucleotide attached via a divalent or trivalent branched linker. one or more GalNAc derivatives] to the subject, thereby treating the subject.

In one aspect, the invention provides a method of treating a subject with a hepatitis B virus (HBV) infection. The method comprises a therapeutically effective amount of a double-stranded RNAi agent [the double-stranded RNAi agent comprises a sense strand and an antisense strand forming a double-stranded region;
The sense strand is 5'-CGUGGUGGUCUUCUCUAAAUU-3' (SEQ ID NO: 37) (or the aforementioned nucleotide sequence and at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% over its entire length; 98%, or 99% identical nucleotide sequence), and the antisense strand comprises 5'-AAUUGAGAGAGAAGUCCACCAGCUU-3' (SEQ ID NO: 38) (or at least 90%, 91%, 92% identical over its entire length to the aforementioned nucleotide sequence); , 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical nucleotide sequence), substantially all of the nucleotides of said sense strand and substantially all of the nucleotides of said antisense strand. are all modified nucleotides, the sense strand is conjugated to a ligand attached at the 3' end, and the ligand is one or more modified nucleotides attached via a divalent or trivalent branched linker. a plurality of GalNAc derivatives] to the subject, thereby treating the subject.

In another aspect, the invention provides a method of treating a subject with a hepatitis B virus (HBV)-related disorder. The method comprises administering a therapeutically effective amount of a double-stranded RNAi agent [the double-stranded RNAi agent comprises a sense strand and an antisense strand forming a double-stranded region, the sense strand being 5'-CGUGGUGGUCUUCUCUAAAUU-3' (sequence No. 37) (or a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical over its entire length to the aforementioned nucleotide sequence) and the antisense strand comprises 5'-AAUUGAGAGAGAAGUCCACCAGCUU-3' (SEQ ID NO: 38) (or the aforementioned nucleotide sequence and at least 90%, 91%, 92%, 93%, 94%, 95%, 96% over its entire length) , 97%, 98%, or 99% identical nucleotide sequences), substantially all of the nucleotides of the sense strand and substantially all of the nucleotides of the antisense strand are modified nucleotides, said sense strand comprising 3 and the ligand is one or more GalNAc derivatives attached via a divalent or trivalent branched linker] to the subject; treating the subject with.

In one aspect, the invention provides a method of treating a subject with a hepatitis B virus (HBV) infection. The method comprises a therapeutically effective amount of a double-stranded RNAi agent [the double-stranded RNAi agent comprises a sense strand and an antisense strand forming a double-stranded region;
The sense strand is 5'-GGUGGACUUCUCUCAAUUUUA-3' (SEQ ID NO: 11) (or the aforementioned nucleotide sequence and at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% over its entire length). , 98%, or 99% identical to nucleotide sequence), and said antisense strand comprises 5'-UAAAAUUGAGAGAGAAGUCCACCAC-3' (SEQ ID NO. 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical nucleotide sequence), substantially all of the nucleotides of the sense strand and of the nucleotides of the antisense strand. Substantially all are modified nucleotides, the sense strand is conjugated to a ligand attached at the 3' end, and the ligand is a nucleotide attached via a divalent or trivalent branched linker. one or more GalNAc derivatives] to the subject, thereby treating the subject.

In another aspect, the invention provides a method of treating a subject with a hepatitis B virus (HBV)-related disorder. The method comprises a therapeutically effective amount of a double-stranded RNAi agent [the double-stranded RNAi agent comprises a sense strand and an antisense strand forming a double-stranded region;
The sense strand is 5'-GGUGGACUUCUCUCAAUUUUA-3' (SEQ ID NO: 11) (or the aforementioned nucleotide sequence and at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% over its entire length). , 98%, or 99% identical nucleotide sequence), and said antisense strand comprises 5'-UAAAAUUGAGAGAGAAGUCCACCAC-3' (SEQ ID NO: 12) (or at least 90%, 91%, over its entire length with said nucleotide sequence); 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical nucleotide sequence), substantially all of the nucleotides of the sense strand and of the nucleotides of the antisense strand. Substantially all are modified nucleotides, the sense strand is conjugated to a ligand attached at the 3' end, and the ligand is a nucleotide attached via a divalent or trivalent branched linker. one or more GalNAc derivatives] to the subject, thereby treating the subject.

In one aspect, the invention provides a method of treating a subject with a hepatitis B virus (HBV) infection. The method comprises a therapeutically effective amount of a double-stranded RNAi agent [the double-stranded RNAi agent comprises a sense strand and an antisense strand forming a double-stranded region;
The sense strand is 5'-GUGUGCACUUCGCUUCACA-3' (SEQ ID NO: 39) (or the aforementioned nucleotide sequence and at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% over its entire length). . 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical nucleotide sequence), substantially all of the nucleotides of the sense strand and of the nucleotides of the antisense strand. Substantially all are modified nucleotides, the sense strand is conjugated to a ligand attached at the 3' end, and the ligand is a nucleotide attached via a divalent or trivalent branched linker. one or more GalNAc derivatives] to the subject, thereby treating the subject.

In another aspect, the invention provides a method of treating a subject with a hepatitis B virus (HBV)-related disorder. The method comprises a therapeutically effective amount of a double-stranded RNAi agent [the double-stranded RNAi agent comprises a sense strand and an antisense strand forming a double-stranded region;
The sense strand is 5'-GUGUGCACUUCGCUUCACA-3' (SEQ ID NO: 39) (or the aforementioned nucleotide sequence and at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% over its entire length). , 98%, or 99% identical nucleotide sequence), and said antisense strand comprises 5'-UGUGAAGCGAAGUGGCACACUU-3' (SEQ ID NO: 40) (or at least 90%, 91%, over its entire length with said nucleotide sequence); 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical nucleotide sequence), substantially all of the nucleotides of the sense strand and of the nucleotides of the antisense strand. Substantially all are modified nucleotides, the sense strand is conjugated to a ligand attached at the 3' end, and the ligand is a nucleotide attached via a divalent or trivalent branched linker. one or more GalNAc derivatives] to the subject, thereby treating the subject.

In one embodiment, all of the nucleotides of said sense strand and all of the nucleotides of said antisense strand contain modifications.

In one embodiment, at least one of the modified nucleotides is a deoxynucleotide, a 3' terminal deoxythymine (dT) nucleotide, a 2'-O-methyl modified nucleotide, a 2'-fluoro modified nucleotide, a 2'-deoxy modified nucleotide, Fixed nucleotides, non-fixed nucleotides, conformationally restricted nucleotides, constrained ethyl nucleotides, non-basic nucleotides, 2'-amino modified nucleotides, 2'-O-allyl modified nucleotides, 2'-C-alkyl modified nucleotides , 2'-hydroxyl modified nucleotide, 2'-methoxyethyl modified nucleotide, 2'-O-alkyl modified nucleotide, morpholino nucleotide, phosphoramidate, nucleotide containing unnatural base, tetrahydropyran modified nucleotide, 1, From the group consisting of 5-anhydrohexitol modified nucleotides, cyclohexenyl modified nucleotides, nucleotides containing a phosphorothioate group, nucleotides containing a methylphosphonate group, nucleotides containing a 5'-phosphate, and nucleotides containing a 5'-phosphate mimetic. selected.

In one embodiment, the 5'-phosphate mimetic is 5'-vinyl phosphate (5'-VP).

In one embodiment, the sense strand comprises 5'-uscsguGfgUfGfGfacuucucuca-3' (SEQ ID NO: 13) and the antisense strand comprises 5'-usGfsagaGfaAfGfuccaCfcAfcgasusu-3' (SEQ ID NO: 14) [wherein A, C, G and U are ribose A, C, G or U; a, g, c and u are 2'-O-methyl (2'-OMe) A, U, C or G; Af , Cf, Gf or Uf is 2'-fluoro A, G, C or U; and s is a phosphorothioate bond].

In another embodiment, the sense strand comprises 5'-uscsguGfgUfGfGfacuucucuca-3' (SEQ ID NO: 15) and the antisense strand comprises 5'-PusGfsagaGfaAfGfuccaCfcAfcgasusu-3' (SEQ ID NO: 16) [wherein A, C , G, and U are ribose A, C, G, or U; a, g, c, and u are 2'-O-methyl (2'-OMe) A, U, C, or G; Af, Cf, Gf or Uf is 2'-fluoro A, G, C or U; and s is a phosphorothioate bond; and P is 5'-phosphate or a 5' phosphate mimetic].

In one embodiment, the sense strand comprises 5'-gsusgcacUfuCfGfCfuucaccucua-3' (SEQ ID NO: 17) and the antisense strand comprises 5'-usAfsgagGfugaagcgAfaGfugcacsusu-3' (SEQ ID NO: 18) [wherein A, C, G and U are ribose A, C, G or U; a, g, c and u are 2'-O-methyl (2'-OMe) A, U, C or G; Af , Cf, Gf or Uf is 2'-fluoro A, G, C or U; and s is a phosphorothioate bond].

In another embodiment, the sense strand comprises 5'-gsusgcacUfuCfGfCfuucaccucua-3' (SEQ ID NO: 19) and the antisense strand comprises 5'-PusAfsgagGfugaagcgAfaGfugcacsusu-3' (SEQ ID NO: 20) [wherein A, C , G, and U are ribose A, C, G, or U; a, g, c, and u are 2'-O-methyl (2'-OMe) A, U, C, or G; Af, Cf, Gf or Uf is 2'-fluoro A, G, C or U; and s is a phosphorothioate bond; and P is 5'-phosphate or a 5' phosphate mimetic].

In one embodiment, the sense strand comprises 5'-csgsugguGfgAfCfUfucucUfCfaauu-3' (SEQ ID NO: 21) and the antisense strand comprises 5'-asAfsuugAfgAfgAfaguCfcAfccagcsasg-3' (SEQ ID NO: 22) [wherein A, C, G and U are ribose A, C, G or U; a, g, c and u are 2'-O-methyl (2'-OMe) A, U, C or G; Af , Cf, Gf or Uf is 2'-fluoro A, G, C or U; and s is a phosphorothioate bond].

In another embodiment, the sense strand comprises 5'-csgsugguGfgAfCfUfucucUfCfaauu-3' (SEQ ID NO: 23) and the antisense strand comprises 5'-PasAfsuugAfgAfgAfaguCfcAfccagcsasg-3' (SEQ ID NO: 24) [wherein A, C , G, and U are ribose A, C, G, or U; a, g, c, and u are 2'-O-methyl (2'-OMe) A, U, C, or G; Af, Cf, Gf or Uf is 2'-fluoro A, G, C or U; and s is a phosphorothioate bond; and P is 5'-phosphate or a 5' phosphate mimetic].

In another embodiment, the sense strand comprises 5'-csgsuggudGgucdTucucuaaauu-3' (SEQ ID NO: 35) and the antisense strand comprises 5'-asdAsuugagagdAagudCcaccagcsusu-3' (SEQ ID NO: 36) [wherein A, C , G, and U are ribose A, C, G, or U; a, g, c, and u are 2'-O-methyl (2'-OMe) A, U, C, or G; dA, dC, dG, and dT are deoxyribose A, C, G, and T; and s is a phosphorothioate bond].

In one embodiment, the sense strand comprises 5'-gsgsuggaCfuUfCfUfcucaAfUfuuua-3' (SEQ ID NO: 25) and the antisense strand comprises 5'-usAfsaaaUfuGfAfgagaAfgUfccaccsasc-3' (SEQ ID NO: 26) [wherein A, C, G and U are ribose A, C, G or U; a, g, c and u are 2'-O-methyl (2'-OMe) A, U, C or G; Af , Cf, Gf or Uf is 2'-fluoro A, G, C or U; and s is a phosphorothioate bond].

In another embodiment, the sense strand comprises 5'-gsgsuggaCfuUfCfUfcucaAfUfuuua-3' (SEQ ID NO: 27) and the antisense strand comprises 5'-PusAfsaaaUfuGfAfgagaAfgUfccaccsasc-3' (SEQ ID NO: 28) [wherein A, C , G, and U are ribose A, C, G, or U; a, g, c, and u are 2'-O-methyl (2'-OMe) A, U, C, or G; Af, Cf, Gf or Uf is 2'-fluoro A, G, C or U; and s is a phosphorothioate bond; and P is 5'-phosphate or a 5' phosphate mimetic].

In another embodiment, the sense strand comprises 5'-gsusguGfcAfCfUfucgcuucaca-3' (SEQ ID NO: 41) and the antisense strand comprises 5'-usGfsugaAfgCfGfaaguGfcAfcacsusu-3' (SEQ ID NO: 42) [wherein A, C , G, and U are ribose A, C, G, or U; a, g, c, and u are 2'-O-methyl (2'-OMe) A, U, C, or G; Af, Cf, Gf or Uf is 2'-fluoro A, G, C or U; and s is a phosphorothioate bond].

である。 In one embodiment, the ligand is

It is.

に示されるリガンドにコンジュゲートされ、式中、ＸはＯ又はＳである。 In one embodiment, the RNAi agent is shown in the schematic diagram below.

where X is O or S.

In one embodiment, the HBV-related disorder is the group consisting of hepatitis D virus infection, delta hepatitis, acute hepatitis B; acute fulminant hepatitis B; chronic hepatitis B; liver fibrosis; end-stage liver disease; hepatocellular carcinoma. selected from.

In one embodiment, the HBV-related disorder is chronic hepatitis and the subject is HBeAg positive. In another embodiment, the HBV-related disorder is chronic hepatitis and the subject is HBeAg negative.

In one aspect, the invention provides a method of treating a subject with a hepatitis B virus (HBV) infection. The method includes administering to a subject a therapeutically effective amount of a composition for inhibiting the expression of hepatitis B virus (HBV) in a cell. This composition comprises: (a) a first double-stranded RNAi agent comprising a first sense strand and a first antisense strand forming a double-stranded region, wherein the nucleotides of the first sense strand are Substantially all and substantially all of the nucleotides of the first antisense strand are modified nucleotides, the first sense strand is conjugated at the 3' end to an attached ligand, and the ligand is a first double-stranded RNAi agent that is one or more GalNAc derivatives linked via a divalent or trivalent branched linker; and (b) a second double-stranded RNAi agent forming a double-stranded region. A second double-stranded RNAi agent comprising a sense strand and a second antisense strand, wherein substantially all of the nucleotides of the second sense strand and substantially all of the nucleotides of the second antisense strand all are modified nucleotides, the second sense strand is conjugated to a ligand attached at the 3' end, and the ligand is a nucleotide attached via a divalent or trivalent branched linker. a second double-stranded RNAi agent that is one or more GalNAc derivatives; the first and second sense strands each independently;
5'-UCGUGGUGGACUUCUCUCA-3' (SEQ ID NO: 5),
5'-GUGCACUUCGCUUCACCUCUA-3' (SEQ ID NO: 7),
5'-CGUGGUGGACUUCUCUCAAUU-3' (SEQ ID NO: 9),
5'-CGUGGUGGCUUCUCUAAAUU-3' (SEQ ID NO: 37),
5'-GGUGGACUUCUCUCAAUUUUA-3' (SEQ ID NO: 11); and 5'-GUGUGCACUUCGCUUCACA-3' (SEQ ID NO: 39); 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical nucleotide sequences), and the first and second antisense strands each hand,
5'-UGAGAGAAGUCCACCACGAUU-3' (SEQ ID NO: 6);
5'-UAGAGGUGAAGCGAAGUGCACUU-3' (SEQ ID NO: 8);
5'-AAUUGAGAGAAGUCCACCAGCAG-3' (SEQ ID NO: 10);
5'-AAUUGAGAGAAGUCCACCAGCUU-3' (SEQ ID NO: 38),
5'-UAAAAUUGAGAGAAGUCCACCAC-3' (SEQ ID NO: 12); 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical nucleotide sequences), thereby treating a subject.

In another aspect, the invention provides a method of treating a subject with a hepatitis B virus (HBV)-related disorder. The method includes administering to a subject a therapeutically effective amount of a composition for inhibiting the expression of hepatitis B virus (HBV) in a cell. This composition comprises: (a) a first double-stranded RNAi agent comprising a first sense strand and a first antisense strand forming a double-stranded region, wherein the nucleotides of the first sense strand are Substantially all and substantially all of the nucleotides of the first antisense strand are modified nucleotides, the first sense strand is conjugated at the 3' end to an attached ligand, and the ligand is a first double-stranded RNAi agent that is one or more GalNAc derivatives linked via a divalent or trivalent branched linker; and (b) a second double-stranded RNAi agent forming a double-stranded region. A second double-stranded RNAi agent comprising a sense strand and a second antisense strand, wherein substantially all of the nucleotides of the second sense strand and substantially all of the nucleotides of the second antisense strand all are modified nucleotides, the second sense strand is conjugated to a ligand attached at the 3' end, and the ligand is a nucleotide attached via a divalent or trivalent branched linker. a second double-stranded RNAi agent that is one or more GalNAc derivatives; the first and second sense strands each independently;
5'-UCGUGGUGGACUUCUCUCA-3' (SEQ ID NO: 5),
5'-GUGCACUUCGCUUCACCUCUA-3' (SEQ ID NO: 7),
5'-CGUGGUGGACUUCUCUCAAUU-3' (SEQ ID NO: 9),
5'-CGUGGUGGCUUCUCUAAAUU-3' (SEQ ID NO: 37),
5'-GGUGGACUUCUCUCAAUUUUA-3' (SEQ ID NO: 11); and 5'-GUGUGCACUUCGCUUCACA-3' (SEQ ID NO: 39); 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical nucleotide sequences), and the first and second antisense strands each hand,
5'-UGAGAGAAGUCCACCACGAUU-3' (SEQ ID NO: 6);
5'-UAGAGGUGAAGCGAAGUGCACUU-3' (SEQ ID NO: 8);
5'-AAUUGAGAGAAGUCCACCAGCAG-3' (SEQ ID NO: 10);
5'-AAUUGAGAGAAGUCCACCAGCUU-3' (SEQ ID NO: 38),
5'-UAAAAUUGAGAGAAGUCCACCAC-3' (SEQ ID NO: 12), and 5'-UGUGAAGCGAAGUGCACACUU-3' (SEQ ID NO: 40); 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical nucleotide sequences), thereby treating a subject.

In one embodiment, all of the nucleotides of the first and second sense strands and all of the nucleotides of the first and second antisense strands include the modification.

In one embodiment, at least one of the modified nucleotides is a deoxynucleotide, a 3' terminal deoxythymine (dT) nucleotide, a 2'-O-methyl modified nucleotide, a 2'-fluoro modified nucleotide, a 2'-deoxy modified nucleotide, Fixed nucleotides, non-fixed nucleotides, conformationally restricted nucleotides, constrained ethyl nucleotides, non-basic nucleotides, 2'-amino modified nucleotides, 2'-O-allyl modified nucleotides, 2'-C-alkyl modified nucleotides , 2'-hydroxyl modified nucleotide, 2'-methoxyethyl modified nucleotide, 2'-O-alkyl modified nucleotide, morpholino nucleotide, phosphoramidate, nucleotide containing unnatural base, tetrahydropyran modified nucleotide, 1, From the group consisting of 5-anhydrohexitol modified nucleotides, cyclohexenyl modified nucleotides, nucleotides containing a phosphorothioate group, nucleotides containing a methylphosphonate group, nucleotides containing a 5'-phosphate, and nucleotides containing a 5'-phosphate mimetic. selected.

In one embodiment, the first and second RNAi agents are
5'-uscsguGfgUfGfGfacuucucuca-3' (SEQ ID NO: 13)
5'-usGfsagaGfaAfGfuccaCfcAfcgasusu-3' (SEQ ID NO: 14);
5'-uscsguGfgUfGfGfacuucucuca-3' (SEQ ID NO: 15)
5'-PusGfsagaGfaAfGfuccaCfcAfcgasusu-3' (SEQ ID NO: 16);
5'-gsusgcacUfuCfGfCfuucaccucua-3' (SEQ ID NO: 17)
5'-usAfsgagGfugaagcgAfaGfugcacsusu-3' (SEQ ID NO: 18);
5'-gsusgcacUfuCfGfCfuucaccucua-3' (SEQ ID NO: 19)
5'-PusAfsgagGfugaagcgAfaGfugcacsusu-3' (SEQ ID NO: 20);
5'-csgsugguGfgAfCfUfucucUfCfauu-3' (SEQ ID NO: 21)
5'-asAfsuugAfgAfgAfaguCfcAfccagcsasg-3' (SEQ ID NO: 22);
5'-csgsugguGfgAfCfUfucucUfCfauu-3' (SEQ ID NO: 23)
5'-PasAfsuugAfgAfgAfaguCfcAfccagcsasg-3' (SEQ ID NO: 24);
5'-csgsuggudGgucdTucucuaaauu-3' (SEQ ID NO: 35)
5'-asdAsuugagagdAagudCcaccagcsusu-3' (SEQ ID NO: 36);
5'-gsgsuggaCfuUfCfUfcucaAfUfuuua-3' (SEQ ID NO: 25)
5'-usAfsaaaUfuGfAfgagaAfgUfccaccsasc-3' (SEQ ID NO: 26);
5'-gsgsuggaCfuUfCfUfcucaAfUfuuua-3' (SEQ ID NO: 27)
5'-PusAfsaaaUfuGfAfgagaAfgUfccaccsasc-3' (SEQ ID NO: 28); and 5'-gsusguGfcAfCfUfucgcuucaca-3' (SEQ ID NO: 41)
5'-usGfsugaAfgCfGfaaguGfcAfcacsusu-3' (SEQ ID NO: 42) [wherein A, C, G, and U are ribose A, C, G, or U; a, g, c, and u are 2'-O -Methyl (2'-OMe) A, U, C, or G; Af, Cf, Gf or Uf is 2'-fluoro A, G, C, or U; dA, dC, dG, and dT are deoxyribose A, C, G, and T; s is a phosphorothioate bond; and P is a 5'-phosphate or a 5' phosphate mimetic.

In one embodiment, the first and second RNAi agents are
5'-uscsguGfgUfGfGfacuucucuca-3' (SEQ ID NO: 15)
5'-PusGfsagaGfaAfGfuccaCfcAfcgasusu-3' (SEQ ID NO: 16); and 5'-csgsugguGfgAfCfUfucucUfCfaauu-3' (SEQ ID NO: 21)
5'-asAfsuugAfgAfgAfaguCfcAfccagcsasg-3' (SEQ ID NO: 22) [wherein A, C, G, and U are ribose A, C, G, or U; a, g, c, and u are 2'-O - methyl (2'-OMe) is A, U, C or G; Af, Cf, Gf or Uf is 2'-fluoro A, G, C or U; s is a phosphorothioate bond; and P is a 5'-phosphate or a 5' phosphate mimetic].

In another embodiment, the first and second RNAi agents are
5'-gsgsuggaCfuUfCfUfcucaAfUfuuua-3' (SEQ ID NO: 25)
5'-usAfsaaaUfuGfAfgagaAfgUfccaccsasc-3' (SEQ ID NO: 26); and 5'-gsusguGfcAfCfUfucgcuucaca-3' (SEQ ID NO: 41)
5'-usGfsugaAfgCfGfaaguGfcAfcacsusu-3' (SEQ ID NO: 42) [wherein A, C, G, and U are ribose A, C, G, or U; a, g, c, and u are 2'-O - methyl (2'-OMe) is A, U, C or G; Af, Cf, Gf or Uf is 2'-fluoro A, G, C or U; s is a phosphorothioate bond; and P is a 5'-phosphate or a 5' phosphate mimetic].

である。 In one embodiment, the ligand is

It is.

に示されるリガンドにコンジュゲートされ、式中、ＸはＯ又はＳである。 In one embodiment, the RNAi agent is shown in the schematic diagram below.

where X is O or S.

In one embodiment, the subject is a human.

In one embodiment, the HBV-related disorder is the group consisting of hepatitis D virus infection, delta hepatitis, acute hepatitis B; acute fulminant hepatitis B; chronic hepatitis B; liver fibrosis; end-stage liver disease; hepatocellular carcinoma. selected from.

In one embodiment, the HBV-related disorder is chronic hepatitis and the subject is HBeAg positive. In another embodiment, the HBV-related disorder is chronic hepatitis and the subject is HBeAg negative.

In one embodiment, the double-stranded RNAi agent is administered at a dose of about 0.01 mg/kg to about 10 mg/kg or about 0.5 mg/kg to about 50 mg/kg.

In one embodiment, the double-stranded RNAi agent is administered at a dose of about 10 mg/kg to about 30 mg/kg. In another embodiment, the double-stranded RNAi agent is administered at a dose of about 3 mg/kg. In one embodiment, the double-stranded RNAi agent is administered at a dose of about 10 mg/kg.

In one embodiment, the double-stranded RNAi agent is administered twice weekly at a dose of about 0.5 mg/kg.

In one embodiment, the double-stranded RNAi agent is administered at a fixed dose of about 50 mg to 200 mg.

In one embodiment, the double-stranded RNAi agent is administered subcutaneously. In another embodiment, the double-stranded RNAi agent is administered intravenously.

In one embodiment, the RNAi agent is administered in two or more doses.

In one embodiment, the RNAi agent is selected from the group consisting of once every 12 hours, once every 24 hours, once every 48 hours, once every 72 hours, and once every 96 hours. Administered at regular intervals.

In one embodiment, the RNAi agent is administered twice weekly. In another embodiment, the RNAi agent is administered biweekly.

In one embodiment, the method of the invention further comprises administering to the subject an additional therapeutic agent.

In one embodiment, the additional therapeutic agent is an antiviral agent, a reverse transcriptase inhibitor, an immunostimulant, a therapeutic vaccine, a viral entry inhibitor, an oligonucleotide that inhibits secretion or release of HbsAg, a capsid inhibitor, a cccDNA inhibitor. selected from the group consisting of drugs, and combinations of any of the foregoing.

In another embodiment, the method of the invention further comprises administering to the subject a reverse transcriptase inhibitor. In yet another embodiment, the method of the invention further comprises administering to the subject a reverse transcriptase inhibitor and an immunostimulant.

In one embodiment, the reverse transcriptase inhibitor is selected from the group consisting of tenofovir disoproxil fumarate (TDF), tenofovir alafenamide, lamivudine, adefovir dipivoxil, entecavir (ETV), telbivudine, and AGX-1009. be done.

In some embodiments, the methods of the invention further include treating hepatitis D virus (HDV) in the subject. The treatment method can include any treatment method known in the art. In certain embodiments, HDV is treated in a subject using one or more of the iRNA agents that target HBV as described herein.

In some embodiments, the methods of the invention further include a method of modulating, eg, reducing, expression of PD-L1. Compositions and methods for reducing the expression of PD-L1 are provided, for example, in WO 2011/127180, the entire contents of which are hereby incorporated by reference.

In one embodiment, the immunostimulant is selected from the group consisting of pegylated interferon α2a (PEG-IFN-α2a), interferon α-2b, recombinant human interleukin-7, and a Toll-like receptor 7 (TLR7) agonist. be done.

In a further aspect, the invention provides a method of treating a subject with a hepatitis B virus (HBV)-related disorder, the method comprising: a therapeutically effective amount of a double-stranded RNAi agent [the double-stranded RNAi agent comprises: comprising a sense strand and an antisense strand forming a double-stranded region,
The sense strand comprises at least 15 contiguous nucleotides that differ by no more than 3 nucleotides from the nucleotide sequence of SEQ ID NO: 29, and the antisense strand comprises at least 15 contiguous nucleotides that differ by no more than 3 nucleotides from the nucleotide sequence of SEQ ID NO: 30. including;
Substantially all of the nucleotides of the sense strand and substantially all of the nucleotides of the antisense strand are modified nucleotides;
The sense strand is conjugated to a ligand attached at the 3' end, and the ligand is one or more GalNAc derivatives attached via a divalent or trivalent branched linker. and thereby treating the subject.

In another aspect, the invention also provides a method of treating a subject with a hepatitis B virus (HBV) infection, which method comprises a composition for inhibiting hepatitis B virus (HBV) expression in a cell. It is a thing,
(a) a first double-stranded RNAi agent comprising a first strand and a first antisense strand forming a double-stranded region,
Substantially all of the nucleotides of the first sense strand and substantially all of the nucleotides of the first antisense strand are modified nucleotides;
The first sense strand is conjugated to a ligand attached at the 3' end, and the ligand is one or more GalNAc derivatives attached via a divalent or trivalent branched linker. a first double-stranded RNAi agent, and
(b) a second double-stranded RNAi agent comprising a second sense strand and a second antisense strand forming a double-stranded region,
Substantially all of the nucleotides of the second sense strand and substantially all of the nucleotides of the second antisense strand are modified nucleotides;
The second sense strand is conjugated to a ligand attached at the 3' end, and the ligand is one or more GalNAc derivatives attached via a divalent or trivalent branched linker. , a second double-stranded RNAi agent [the first sense strand comprises at least 15 contiguous nucleotides that differ by no more than three nucleotides from the nucleotide sequence of SEQ ID NO: 1, and the first antisense strand comprises a nucleotide sequence of SEQ ID NO: 1; comprises at least 15 contiguous nucleotides that differ by no more than 3 nucleotides from the nucleotide sequence of 2;
The second sense strand comprises at least 15 contiguous nucleotides that differ by no more than 3 nucleotides from the nucleotide sequence of SEQ ID NO: 29, and the second antisense strand comprises at least 15 contiguous nucleotides that differ from the nucleotide sequence of SEQ ID NO: 30 by no more than 3 nucleotides. at least 15 different contiguous nucleotides], thereby treating the subject.

In some embodiments, the first sense strand is
5'-UCGUGGUGGACUUCUCUCA-3' (SEQ ID NO: 5),
5'-GUGCACUUCGCUUCACCUCUA-3' (SEQ ID NO: 7),
5'-CGUGGUGGACUUCUCUCAAUU-3' (SEQ ID NO: 9),
5'-CGUGGUGGCUUCUCUAAAUU-3' (SEQ ID NO: 37)
5'-GGUGGACUUCUCUCAAUUUUA-3' (SEQ ID NO: 11), and 5'-GUGUGCACUUCGCUUCACA-3' (SEQ ID NO: 39) , 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical nucleotide sequence), and the second antisense strand comprises:
5'-UGAGAGAAGUCCACCACGAUU-3' (SEQ ID NO: 6);
5'-UAGAGGUGAAGCGAAGUGCACUU-3' (SEQ ID NO: 8);
5'-AAUUGAGAGAAGUCCACCAGCAG-3' (SEQ ID NO: 10);
5'-AAUUGAGAGAAGUCCACCAGCUU-3' (SEQ ID NO: 38);
5'-UAAAAUUGAGAGAAGUCCACCAC-3' (SEQ ID NO: 12); and 5'-UGUGAAGCGAAGUGCACACUU-3' (SEQ ID NO: 40); , 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical nucleotide sequences).

In some embodiments, all of the nucleotides of the sense strand and all of the nucleotides of the antisense strand contain modifications.

In certain embodiments, at least one of the modified nucleotides is a deoxynucleotide, a 3' terminal deoxythymine (dT) nucleotide, a 2'-O-methyl modified nucleotide, a 2'-fluoro modified nucleotide, a 2'-deoxy modified nucleotide, Fixed nucleotides, non-fixed nucleotides, conformationally restricted nucleotides, constrained ethyl nucleotides, non-basic nucleotides, 2'-amino modified nucleotides, 2'-O-allyl modified nucleotides, 2'-C-alkyl modified nucleotides , 2'-hydroxyl modified nucleotide, 2'-methoxyethyl modified nucleotide, 2'-O-alkyl modified nucleotide, morpholino nucleotide, phosphoramidate, nucleotide containing unnatural base, tetrahydropyran modified nucleotide, 1, From the group consisting of 5-anhydrohexitol modified nucleotides, cyclohexenyl modified nucleotides, nucleotides containing a phosphorothioate group, nucleotides containing a methylphosphonate group, nucleotides containing a 5'-phosphate, and nucleotides containing a 5'-phosphate mimetic. selected.

である。 In some embodiments, the ligand is

It is.

に示されるリガンドにコンジュゲートされ、式中、ＸはＯ又はＳである。 In a specific embodiment, the RNAi agent is shown in the schematic diagram below.

where X is O or S.

In certain embodiments, double-stranded RNAi agents and compositions provided herein are used to treat HDV infection and/or HDV-related disorders.

Accordingly, the present invention provides methods of inhibiting hepatitis D virus (HDV) replication in cells. The method includes the steps of: (a) contacting a cell with a double-stranded RNAi agent, composition, vector, or pharmaceutical composition provided herein; , maintaining the HBV gene mRNA transcript for a sufficient period of time to effect degradation, thereby inhibiting HDV replication in the cell.

In certain embodiments, the cell is within the subject's body. In certain embodiments, the subject is a human.

The invention further provides a method of reducing hepatitis D virus (HDV) antigen levels in a subject infected with HDV. The method includes administering to a subject a therapeutically effective amount of a double-stranded RNAi agent, composition, vector, or pharmaceutical composition provided herein, thereby detecting the subject's HDV antigen, such as S-HDAg or L- reducing the level of HDAg.

The present invention also provides methods of reducing hepatitis D virus (HDV) viral load in a subject infected with HDV. The method comprises administering to a subject a therapeutically effective amount of a double-stranded RNAi agent, composition, vector, or pharmaceutical composition provided herein, thereby reducing the HDV viral load in the subject. .

The present invention also provides a method of treating a subject with a hepatitis D virus (HDV) infection, which method comprises using a double-stranded RNAi agent, composition, vector, or pharmaceutical composition provided herein. administering to the subject a therapeutically effective amount of, thereby treating the subject.

In certain embodiments, double-stranded RNAi agents include a sense strand and an antisense strand that form a double-stranded region. The sense and antisense strands can be selected from the following RNAi agents, where the sense strand comprises 5'-UCGUGGUGGACUUCUCUCA-3' (SEQ ID NO: 5) and the antisense strand comprises 5'-UGAGAGAAGUCCACCACGAUU-3 ' (SEQ ID NO: 6); the sense strand includes 5'-GUGCACUUCGCUUCACCUCUA-3' (SEQ ID NO: 7), and the antisense strand includes 5'-UAGAGGUGAAGCGAAGUGCACUU-3' (SEQ ID NO: 8); sense The strand comprises 5'-CGUGGUGGACUUCUCAAUU-3' (SEQ ID NO: 9) and the antisense strand comprises 5'-AAUUGAGAGAGAAGUCCACCAGCAG-3' (SEQ ID NO: 10); No. 37), and the antisense strand comprises 5'-AAUUGAGAGAAGAAGUCCACCAGCUU-3' (SEQ ID NO: 38); or the sense strand comprises 5'-GUGUGCACUUCGCUUCACA-3' (SEQ ID NO: 39) and the antisense strand comprises 5'-UGUGAAGCGAAGUGCACACUU-3' (SEQ ID NO: 40), wherein substantially all of the nucleotides of the sense strand and substantially all of the nucleotides of the antisense strand are modified nucleotides, the sense strand is conjugated at the 3' end to a bound ligand; is one or more GalNAc derivatives attached via a divalent or trivalent branched linker, thereby treating a subject.

In certain embodiments, all of the nucleotides of the sense strand and all of the nucleotides of the antisense strand contain modifications. In certain embodiments, at least one of the modified nucleotides is a deoxynucleotide, a 3' terminal deoxythymine (dT) nucleotide, a 2'-O-methyl modified nucleotide, a 2'-fluoro modified nucleotide, a 2'-deoxy modified nucleotide, Fixed nucleotides, non-fixed nucleotides, conformationally restricted nucleotides, constrained ethyl nucleotides, non-basic nucleotides, 2'-amino modified nucleotides, 2'-O-allyl modified nucleotides, 2'-C-alkyl modified nucleotides , 2'-hydroxyl modified nucleotide, 2'-methoxyethyl modified nucleotide, 2'-O-alkyl modified nucleotide, morpholino nucleotide, phosphoramidate, nucleotide containing unnatural base, tetrahydropyran modified nucleotide, 1, From the group consisting of 5-anhydrohexitol modified nucleotides, cyclohexenyl modified nucleotides, nucleotides containing a phosphorothioate group, nucleotides containing a methylphosphonate group, nucleotides containing a 5'-phosphate, and nucleotides containing a 5'-phosphate mimetic. selected. In certain embodiments, the 5'-phosphate mimetic is 5'-vinyl phosphate (5'-VP).

In certain embodiments, the sense strand comprises 5'-uscsguGfgUfGfGfacuucucuca-3' (SEQ ID NO: 13) and the antisense strand comprises 5'-usGfsagaGfaAfGfuccaCfcAfcgasusu-3' (SEQ ID NO: 14) [wherein A, C , G, and U are ribose A, C, G, or U; a, g, c, and u are 2'-O-methyl (2'-OMe) A, U, C, or G; Af, Cf, Gf or Uf is 2'-fluoro A, G, C or U; and s is a phosphorothioate bond].

In certain embodiments, the sense strand comprises 5'-uscsguGfgUfGfGfacuucucuca-3' (SEQ ID NO: 15) and the antisense strand comprises 5'-PusGfsagaGfaAfGfuccaCfcAfcgasusu-3' (SEQ ID NO: 16) [wherein A, C , G, and U are ribose A, C, G, or U; a, g, c, and u are 2'-O-methyl (2'-OMe) A, U, C, or G; Af, Cf, Gf or Uf is 2'-fluoro A, G, C or U; and s is a phosphorothioate bond; and P is 5'-phosphate or a 5' phosphate mimetic].

In certain embodiments, the sense strand comprises 5'-gsusgcacUfuCfGfCfuucaccucua-3' (SEQ ID NO: 17) and the antisense strand comprises 5'-usAfsgagGfugaagcgAfaGfugcacsusu-3' (SEQ ID NO: 18) [wherein A, C , G, and U are ribose A, C, G, or U; a, g, c, and u are 2'-O-methyl (2'-OMe) A, U, C, or G; Af, Cf, Gf or Uf is 2'-fluoro A, G, C or U; and s is a phosphorothioate bond].

In certain embodiments, the sense strand comprises 5'-gsusgcacUfuCfGfCfuucaccucua-3' (SEQ ID NO: 19) and the antisense strand comprises 5'-PusAfsgagGfugaagcgAfaGfugcacsusu-3' (SEQ ID NO: 20) [wherein A, C , G, and U are ribose A, C, G, or U; a, g, c, and u are 2'-O-methyl (2'-OMe) A, U, C, or G; Af, Cf, Gf or Uf is 2'-fluoro A, G, C or U; and s is a phosphorothioate bond; and P is 5'-phosphate or a 5' phosphate mimetic].

In certain embodiments, the sense strand comprises 5'-csgsugguGfgAfCfUfucucUfCfaauu-3' (SEQ ID NO: 21) and the antisense strand comprises 5'-AfsuugAfgAfgAfaguCfcAfccagcsasg-3' (SEQ ID NO: 22) [wherein A, C , G, and U are ribose A, C, G, or U; a, g, c, and u are 2'-O-methyl (2'-OMe) A, U, C, or G; Af, Cf, Gf or Uf is 2'-fluoro A, G, C or U; and s is a phosphorothioate bond].

In certain embodiments, the sense strand comprises 5'-csgsugguGfgAfCfUfucucUfCfaauu-3' (SEQ ID NO: 23) and the antisense strand comprises 5'-PasAfsuugAfgAfgAfaguCfcAfccagcsasg-3' (SEQ ID NO: 24) [wherein A, C , G, and U are ribose A, C, G, or U; a, g, c, and u are 2'-O-methyl (2'-OMe) A, U, C, or G; Af, Cf, Gf or Uf is 2'-fluoro A, G, C or U; and s is a phosphorothioate bond; and P is 5'-phosphate or a 5' phosphate mimetic].

In certain embodiments, the sense strand comprises 5'-csgsuggudGgucdTucucuaaauu-3' (SEQ ID NO: 35) and the antisense strand comprises 5'-asdAsuugagagdAagudCcaccagcsusu-3' (SEQ ID NO: 36) [wherein A, C , G, and U are ribose A, C, G, or U; a, g, c, and u are 2'-O-methyl (2'-OMe) A, U, C, or G; dA, dC, dG, and dT are deoxyribose A, C, G, and T; and s is a phosphorothioate bond].

In certain embodiments, the sense strand comprises 5'-gsgsuggaCfuUfCfUfcucaAfUfuuua-3' (SEQ ID NO: 25) and the antisense strand comprises 5'-usAfsaaaUfuGfAfgagaAfgUfccaccsasc-3' (SEQ ID NO: 26) [wherein A, C , G, and U are ribose A, C, G, or U; a, g, c, and u are 2'-O-methyl (2'-OMe) A, U, C, or G; Af, Cf, Gf or Uf is 2'-fluoro A, G, C or U; and s is a phosphorothioate bond].

In certain embodiments, the sense strand comprises 5'-gsgsuggaCfuUfCfUfcucaAfUfuuua-3' (SEQ ID NO: 27) and the antisense strand comprises 5'-PusAfsaaaUfuGfAfgagaAfgUfccaccsasc-3' (SEQ ID NO: 28) [wherein A, C , G, and U are ribose A, C, G, or U; a, g, c, and u are 2'-O-methyl (2'-OMe) A, U, C, or G; Af, Cf, Gf or Uf is 2'-fluoro A, G, C or U; and s is a phosphorothioate bond; and P is 5'-phosphate or a 5' phosphate mimetic].

In certain embodiments, the sense strand comprises 5'-gsusguGfcAfCfUfucgcuucaca-3' (SEQ ID NO: 41) and the antisense strand comprises 5'-usGfsugaAfgCfGfaaguGfcAfcacsusu-3' (SEQ ID NO: 42) [wherein A, C , G, and U are ribose A, C, G, or U; a, g, c, and u are 2'-O-methyl (2'-OMe) A, U, C, or G; Af, Cf, Gf or Uf is 2'-fluoro A, G, C or U; and s is a phosphorothioate bond].

である。 In certain embodiments, the ligand is

It is.

に示されるリガンドにコンジュゲートされ、式中、ＸはＯ又はＳである。 In certain embodiments, the RNAi agent is shown in the schematic diagram below.

where X is O or S.

The present invention provides a method of treating a subject with a hepatitis D virus (HDV) infection. The method provides a composition for inhibiting hepatitis B virus (HBV) expression in a cell, the composition comprising: (a) a first sense strand and a first antisense strand forming a double-stranded region; a first double-stranded RNAi agent, wherein substantially all of the nucleotides of the first sense strand and substantially all of the nucleotides of the first antisense strand are modified nucleotides; , and the ligand is one or more GalNAc derivatives attached via a divalent or trivalent branched linker. and (b) a second double-stranded RNAi agent comprising a second sense strand and a second antisense strand forming a double-stranded region, the second double-stranded RNAi agent comprising a nucleotide substance of the second sense strand; all and substantially all of the nucleotides of the second antisense strand are modified nucleotides, the second sense strand is conjugated to a ligand attached at the 3' end, and the ligand is divalent or a second double-stranded RNAi agent that is one or more GalNAc derivatives linked via a trivalent branched linker [the first and second sense strands each independently
5'-UCGUGGUGGACUUCUCUCA-3' (SEQ ID NO: 5),
5'-GUGCACUUCGCUUCACCUCUA-3' (SEQ ID NO: 7),
5'-CGUGGUGGACUUCUCUCAAUU-3' (SEQ ID NO: 9),
5'-CGUGGUGGCUUCUCUAAAUU-3' (SEQ ID NO: 37),
5'-GGUGGACUUCUCUCAAUUUUA-3' (SEQ ID NO: 11), and 5'-GUGUGCACUUCGCUUCACA-3' (SEQ ID NO: 39), and the first and second antisense strands each Independently,
5'-UGAGAGAAGUCCACCACGAUU-3' (SEQ ID NO: 6);
5'-UAGAGGUGAAGCGAAGUGCACUU-3' (SEQ ID NO: 8);
5'-AAUUGAGAGAAGUCCACCAGCAG-3' (SEQ ID NO: 10);
5'-AAUUGAGAGAAGUCCACCAGCUU-3' (SEQ ID NO: 38),
5'-UAAAAUUGAGAGAAGUCCACCAC-3' (SEQ ID NO: 12), and 5'-UGUGAAGCGAAGUGCACACUU-3' (SEQ ID NO: 40)]. , thereby treating the subject.

In certain embodiments, all of the nucleotides of the first and second sense strands and all of the nucleotides of the first and second antisense strands include the modification. In certain embodiments, at least one of the modified nucleotides is a deoxynucleotide, a 3' terminal deoxythymine (dT) nucleotide, a 2'-O-methyl modified nucleotide, a 2'-fluoro modified nucleotide, a 2'-deoxy modified nucleotide, Fixed nucleotides, non-fixed nucleotides, conformationally restricted nucleotides, constrained ethyl nucleotides, non-basic nucleotides, 2'-amino modified nucleotides, 2'-O-allyl modified nucleotides, 2'-C-alkyl modified nucleotides , 2'-hydroxyl modified nucleotide, 2'-methoxyethyl modified nucleotide, 2'-O-alkyl modified nucleotide, morpholino nucleotide, phosphoramidate, nucleotide containing unnatural base, tetrahydropyran modified nucleotide, 1, From the group consisting of 5-anhydrohexitol modified nucleotides, cyclohexenyl modified nucleotides, nucleotides containing a phosphorothioate group, nucleotides containing a methylphosphonate group, nucleotides containing a 5'-phosphate, and nucleotides containing a 5'-phosphate mimetic. selected.

In certain embodiments, the first and second RNAi agents are of the group:
5'-uscsguGfgUfGfGfacuucucuca-3' (SEQ ID NO: 13)
5'-usGfsagaGfaAfGfuccaCfcAfcgasusu-3' (SEQ ID NO: 14);
5'-uscsguGfgUfGfGfacuucucuca-3' (SEQ ID NO: 15)
5'-PusGfsagaGfaAfGfuccaCfcAfcgasusu-3' (SEQ ID NO: 16);
5'-gsusgcacUfuCfGfCfuucaccucua-3' (SEQ ID NO: 17)
5'-usAfsgagGfugaagcgAfaGfugcacsusu-3' (SEQ ID NO: 18);
5'-gsusgcacUfuCfGfCfuucaccucua-3' (SEQ ID NO: 19)
5'-PusAfsgagGfugaagcgAfaGfugcacsusu-3' (SEQ ID NO: 20);
5'-csgsugguGfgAfCfUfucucUfCfauu-3' (SEQ ID NO: 21)
5'-asAfsuugAfgAfgAfaguCfcAfccagcsasg-3' (SEQ ID NO: 22);
5'-csgsugguGfgAfCfUfucucUfCfauu-3' (SEQ ID NO: 23)
5'-PasAfsuugAfgAfgAfaguCfcAfccagcsasg-3' (SEQ ID NO: 24);
5'-csgsuggudGgucdTucucuaaauu-3' (SEQ ID NO: 35)
5'-asdAsuugagagdAagudCcaccagcsusu-3' (SEQ ID NO: 36);
5'-gsgsuggaCfuUfCfUfcucaAfUfuuua-3' (SEQ ID NO: 25)
5'-usAfsaaaUfuGfAfgagaAfgUfccaccsasc-3' (SEQ ID NO: 26);
5'-gsgsuggaCfuUfCfUfcucaAfUfuuua-3' (SEQ ID NO: 27)
5'-PusAfsaaaUfuGfAfgagaAfgUfccaccsasc-3' (SEQ ID NO: 28); and 5'-gsusguGfcAfCfUfucgcuucaca-3' (SEQ ID NO: 41)
5'-usGfsugaAfgCfGfaaguGfcAfcacsusu-3' (SEQ ID NO: 42) [wherein A, C, G, and U are ribose A, C, G, or U; a, g, c, and u are 2'-O -Methyl (2'-OMe) A, U, C, or G; Af, Cf, Gf or Uf is 2'-fluoro A, G, C, or U; dA, dC, dG, and dT are deoxyribose A, C, G, and T; s is a phosphorothioate bond; and P is a 5'-phosphate or a 5' phosphate mimetic].

In certain embodiments, the first and second RNAi agents are
5'-uscsguGfgUfGfGfacuucucuca-3' (SEQ ID NO: 15)
5'-PusGfsagaGfaAfGfuccaCfcAfcgasusu-3' (SEQ ID NO: 16); and 5'-csgsugguGfgAfCfUfucucUfCfaauu-3' (SEQ ID NO: 21)
5'-asAfsuugAfgAfgAfaguCfcAfccagcsasg-3' (SEQ ID NO: 22) [wherein A, C, G, and U are ribose A, C, G, or U; a, g, c, and u are 2'-O - methyl (2'-OMe) is A, U, C or G; Af, Cf, Gf or Uf is 2'-fluoro A, G, C or U; s is a phosphorothioate bond; and P is a 5'-phosphate or a 5' phosphate mimetic].

In certain embodiments, the first and second RNAi agents are
5'-gsgsuggaCfuUfCfUfcucaAfUfuuua-3' (SEQ ID NO: 25)
5'-usAfsaaaUfuGfAfgagaAfgUfccaccsasc-3' (SEQ ID NO: 26); and 5'-gsusguGfcAfCfUfucgcuucaca-3' (SEQ ID NO: 41)
5'-usGfsugaAfgCfGfaaguGfcAfcacsusu-3' (SEQ ID NO: 42) [wherein A, C, G, and U are ribose A, C, G, or U; a, g, c, and u are 2'-O - methyl (2'-OMe) is A, U, C or G; Af, Cf, Gf or Uf is 2'-fluoro A, G, C or U; s is a phosphorothioate bond; and P is a 5'-phosphate or a 5' phosphate mimetic].

である。 In certain embodiments, the ligand is

It is.

に示されるリガンドにコンジュゲートされ、式中、ＸはＯ又はＳである。 In certain embodiments, the RNAi agent is shown in the schematic diagram below.

where X is O or S.

In certain embodiments, the subject is a human.

In certain embodiments, double-stranded RNAi agents are administered at a dose of about 0.01 mg/kg to about 10 mg/kg or about 0.5 mg/kg to about 50 mg/kg. In certain embodiments, double-stranded RNAi agents are administered at a dose of about 10 mg/kg to about 30 mg/kg. In certain embodiments, the double-stranded RNAi agent is administered at a dose of about 3 mg/kg. In certain embodiments, double-stranded RNAi agents are administered at a dose of about 10 mg/kg. In certain embodiments, the double-stranded RNAi agent is administered twice weekly at a dose of about 0.5 mg/kg. In certain embodiments, the double-stranded RNAi agent is administered at a fixed dose of about 50 mg to 200 mg.

In certain embodiments, double-stranded RNAi agents are administered subcutaneously.

In certain embodiments, double-stranded RNAi agents are administered intravenously.

In certain embodiments, the RNAi agent is administered in two or more doses. In certain embodiments, the RNAi agent is selected from the group consisting of once every 12 hours, once every 24 hours, once every 48 hours, once every 72 hours, and once every 96 hours. Administered at regular intervals. In certain embodiments, the RNAi agent is administered twice weekly. In certain embodiments, the RNAi agent is administered biweekly. In certain embodiments, the RNAi agent is administered once a month. In certain embodiments, the RNAi agent is administered once every other month. In certain embodiments, the RNAi agent is administered once every three months.

In certain embodiments, the RNAi agent is administered to the subject in conjunction with an additional therapeutic agent. Additional therapeutic agents include, for example, antiviral agents, reverse transcriptase inhibitors, immunostimulants, therapeutic vaccines, viral entry inhibitors, oligonucleotides that inhibit HbsAg secretion or release, capsid inhibitors, covalently closed cyclic (ccc) HBV DNA inhibitors, and combinations of any of the foregoing.

In certain embodiments, the additional agent is a reverse transcriptase inhibitor. In certain embodiments, the additional agents are reverse transcriptase inhibitors and immunostimulants. Exemplary reverse transcriptase inhibitors include tenofovir disoproxil fumarate (TDF), tenofovir alafenamide, lamivudine, adefovir dipivoxil, entecavir (ETV), telbivudine, and AGX-1009. Exemplary immunostimulants include pegylated interferon alpha 2a (PEG-IFN- alpha 2a), interferon alpha-2b, recombinant human interleukin-7, and Toll-like receptor 7 (TLR7) agonists.

The invention further provides a method of treating a subject with hepatitis D virus (HDV) infection, the method comprising: a composition for inhibiting hepatitis B virus (HBV) expression in a cell; (a) a first double-stranded RNAi agent comprising a first strand and a first antisense strand forming a double-stranded region, the first double-stranded RNAi agent comprising substantially all of the nucleotides of the first sense strand and the first antisense strand; Substantially all of the nucleotides of the antisense strand of the first sense strand are modified nucleotides, the first sense strand is conjugated to a ligand attached at the 3' end, and the ligand is a divalent or trivalent branched chain. a first double-stranded RNAi agent that is one or more GalNAc derivatives linked via a linker; and (b) a second sense strand and a second antisense strand forming a double-stranded region. a second double-stranded RNAi agent comprising: substantially all of the nucleotides of the second sense strand and substantially all of the nucleotides of the second antisense strand are modified nucleotides; The chain is conjugated to a ligand attached at the 3' end, and the ligand is a second GalNAc derivative, which is one or more GalNAc derivatives attached via a divalent or trivalent branched linker. Double-stranded RNAi agents [the first sense strand comprises at least 15 contiguous nucleotides that differ by no more than three nucleotides from the nucleotide sequence of SEQ ID NO: 1, and the first antisense strand differs from the nucleotide sequence of SEQ ID NO: 2; the second sense strand comprises at least 15 consecutive nucleotides that differ by no more than 3 nucleotides from the nucleotide sequence of SEQ ID NO: 29, and the second antisense strand comprises at least 15 contiguous nucleotides that differ by no more than 3 nucleotides from the nucleotide sequence of SEQ ID NO: , comprising at least 15 contiguous nucleotides that differ by no more than 3 nucleotides from the nucleotide sequence of SEQ ID NO: 30], thereby treating the subject.

In certain embodiments, the first sense strand is
5'-UCGUGGUGGACUUCUCUCA-3' (SEQ ID NO: 5),
5'-GUGCACUUCGCUUCACCUCUA-3' (SEQ ID NO: 7),
5'-CGUGGUGGACUUCUCUCAAUU-3' (SEQ ID NO: 9),
5'-CGUGGUGGCUUCUCUAAAUU-3' (SEQ ID NO: 37),
5'-GGUGGACUUCUCUCAAUUUUA-3' (SEQ ID NO: 11), and 5'-GUGUGCACUUCGCUUCACA-3' (SEQ ID NO: 39), and the second antisense strand comprises:
5'-UGAGAGAAGUCCACCACGAUU-3' (SEQ ID NO: 6);
5'-UAGAGGUGAAGCGAAGUGCACUU-3' (SEQ ID NO: 8);
5'-AAUUGAGAGAAGUCCACCAGCAG-3' (SEQ ID NO: 10);
5'-AAUUGAGAGAAGUCCACCAGCUU-3' (SEQ ID NO: 38),
5'-UAAAAUUGAGAGAAGUCCACCAC-3' (SEQ ID NO: 12), and 5'-UGUGAAGCGAAGUGCACACUU-3' (SEQ ID NO: 40).

In certain embodiments, all of the nucleotides of the sense strand and all of the nucleotides of the antisense strand contain modifications. In certain embodiments, the additional agent is a dexoy-nucleotide, a 3' terminal deoxythymine (dT) nucleotide, a 2'-O-methyl modified nucleotide, a 2'-fluoro modified nucleotide, a 2'-deoxy modified nucleotide. , fixed nucleotide, non-fixed nucleotide, conformationally restricted nucleotide, constrained ethyl nucleotide, non-basic nucleotide, 2'-amino modified nucleotide, 2'-O-allyl modified nucleotide, 2'-C-alkyl modified Nucleotides, 2'-hydroxyl modified nucleotides, 2'-methoxyethyl modified nucleotides, 2'-O-alkyl modified nucleotides, morpholinonucleotides, phosphoramidates, nucleotides containing unnatural bases, tetrahydropyran modified nucleotides, 1 , 5-anhydrohexitol modified nucleotides, cyclohexenyl modified nucleotides, nucleotides containing a phosphorothioate group, nucleotides containing a methylphosphonate group, nucleotides containing a 5'-phosphate, and nucleotides containing a 5'-phosphate mimetic. at least one modified nucleotide selected from

である。 In certain embodiments, the ligand is

It is.

に示されるリガンドにコンジュゲートされ、式中、ＸはＯ又はＳである。 In certain embodiments, the RNAi agent is shown in the schematic diagram below.

where X is O or S.

The invention is further illustrated by the following detailed description and drawings.

The structure of the approximately 3.2 kb double-stranded HBV genome is schematically shown. Replication of the HBV genome occurs through RNA intermediates, resulting in four overlapping viral transcripts encoding seven viral proteins (pre-S1, pre-S2, S, P, X, pre-C and C) that are translated across three reading frames. (approximately 3.5 kb transcript, approximately 2.4 kb transcript, approximately 2.1 kb transcript, and approximately 0.7 kb transcript). Figure 2 is a graph showing the log reduction in HBsAg serum levels normalized to pre-dose HBsAg serum levels after administration of a single 3 mg/kg dose of an indicator iRNA agent. Figure 2 is a graph showing the log reduction in HBsAg serum levels normalized to pre-dose HBsAg serum levels after administration of a single 3 mg/kg dose of an indicator iRNA agent. Figure 2 is a graph showing the percentage of pre-dose HBsAg remaining on days 5 and 10 after administration of a single 3 mg/kg dose of an indicator iRNA agent. Figure 4 also shows the percentage of HBsAG remaining 10 days after administration compared to the percentage of HBsAG remaining at 10 days after administration in animals administered 3 mg/kg control dsRNA targeting mouse/rat transthyretin (mrTTR). The percentage of HBsAG remaining in the eye is also shown. Figure 2 is a graph showing the log reduction in HBsAg serum levels normalized to pre-dose HBsAg serum levels after administration of a single 3 mg/kg dose of AD-65403. Standardized reduction in HBsAg serum levels normalized to pre-dose HBsAg serum levels after administration of a single subcutaneous 0.3 mg/kg, 1 mg/kg, 3 mg/kg, or 9 mg/kg dose of AD-66810 Figure 2 is a graph shown on a linear scale. Log10 reduction in HBsAg serum levels normalized to pre-dose HBsAg serum levels after administration of a single subcutaneous 0.3 mg/kg, 1 mg/kg, 3 mg/kg, or 9 mg/kg dose of AD-66810 It is a graph shown on a scale. Figure 2 is a graph showing the decrease in HBsAg plasma levels on a log10 scale, normalized to pre-dose HBsAg plasma levels, after administration of three weekly subcutaneous 3 mg/kg doses of AD-66810.

The present invention provides iRNA compositions that produce RNA-induced silencing complex (RISC)-mediated cleavage of RNA transcripts of hepatitis B virus (HBV) genes. The gene may be located within a cell, eg, within a cell within the body of a subject, such as a human. Using these iRNAs, it is possible to target and degrade the mRNA of the corresponding gene (HBV gene) in mammals.

The RNAi agents of the invention are designed to target regions within the HBV genome that are conserved across all eight serotypes of HBV. In addition, the RNAi agents of the present invention inhibit all stages of the HBV life cycle, such as viral replication, assembly, secretion, and secretion of subviral antigens, by inhibiting the expression of two or more HBV genes. is designed to. In particular, since polycistronic overlapping RNAs are generated when the HBV genome is transcribed, the RNAi agents of the present invention that target a single HBV gene will significantly inhibit the expression of most or all HBV transcripts. It turns out. For example, since the HBV genome is transcribed into a single mRNA, an RNAi agent of the invention that targets the S gene will inhibit not only S gene expression, but also the expression of "downstream" polymerase genes. Additionally, the RNAi agents of the invention inhibit HBV viral replication by targeting the HBV structural genes and the HBV X gene, thereby allowing the subject's immune system to detect and respond to the presence of HBsAg. , is designed to be able to produce anti-HBV antibodies and eliminate HBV infection. Without intending to be limited by theory, it is believed that combinations or subcombinations of the foregoing properties and specific target sites and/or specific modifications of these RNAi agents may provide efficacy for the RNAi agents of the present invention. , are believed to confer improved stability, safety, efficacy, and persistence.

Using in vitro and in vivo assays, we demonstrated that iRNAs targeting HBV genes can potently mediate RNAi and result in significant inhibition of the expression of two or more HBV genes. The inventors also demonstrated that the RNAi agents of the invention are highly stable in the cytoplasm and lysosomes. Accordingly, methods and compositions comprising these iRNAs are useful for treating subjects with HBV infection and/or HBV-related diseases, such as chronic hepatitis B (CHB).

Accordingly, the present invention also provides a method for treating subjects with disorders that may benefit from inhibiting or reducing the expression of HBV genes, such as HBV-associated diseases such as chronic hepatitis B virus infection (CHB), by reducing the RNA transcription of HBV genes. Also provided are methods of treatment using iRNA compositions that produce RNA-induced silencing complex (RISC)-mediated cleavage of a substance.

In particular, very low dosages of the iRNA of the invention can specifically and efficiently mediate RNA interference (RNAi), resulting in significant inhibition of the expression of the corresponding gene (HBV gene).

The iRNA of the present invention has a length of about 30 nucleotides or less, for example, 15-30, 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18- 23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21- A region having a length of 30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 nucleotides, which comprises at least one of the mRNA transcripts of the HBV gene. It contains an RNA strand (antisense strand) that has a region that is substantially complementary to a portion thereof.

The following detailed description describes how to make and use compositions containing iRNA to inhibit the expression of HBV genes, and how one can benefit from inhibiting and/or reducing the expression of HBV genes. Compositions, uses, and methods for treating subjects with diseases and disorders are disclosed.

I. Definitions In order that the present invention may be more easily understood, some terms are first defined. Furthermore, it is noted that whenever a value or range of values is recited for a variable, intermediate values and ranges of the recited values are also intended to be part of the invention.

The articles "a" and "an" are used herein to refer to one or more (i.e., at least one) of the grammatical object of the article. . By way of example, "an element" means one element or more than one element, eg, a plurality of elements.

The term "including" is used herein to mean the phrase "including but not limited to," and may be used interchangeably with this phrase. be done.

The term "or" is used herein to mean and is used interchangeably with the term "and/or," unless the context clearly indicates otherwise.

As used herein, the term "hepatitis B virus" used synonymously with the term "HBV" refers to a well-known non-cytopathic hepatotropic DNA virus belonging to the Hepadnaviridae family. . The HBV genome is a partially double-stranded circular DNA with overlapping reading frames (see, eg, Figure 1).

There are four known genes encoded by the HBC genome, designated C, X, P, and S. The core protein is encoded by gene C (HBcAg). Hepatitis B antigen (HBeAg) is produced by proteolytic processing of precore (preC) protein. DNA polymerase is encoded by gene P. Gene S is a gene encoding a surface antigen (HBsAg). The HBsAg gene is one long open reading frame but contains three in-frame "start" (ATG) codons that divide the gene into three sections, pre-S1, pre-S2, and S. Because of the multiple start codons, three different sizes of polypeptides are produced, called large, middle, and small (pre-S1+pre-S2+S, pre-S2+S, or S). Although the function of the nonstructural protein encoded by gene encodes a decoy protein that allows evasion.

Proteins encoded by the HBV genome include envelope protein - i) small, hepatitis B surface antigen (HBsAg); ii) middle - preS2+HBsAg; iii) large - preSl+preS2+HBsAg; nucleocapsid protein, hepatitis B core antigen. (HBcAg). Hepatitis B e antigen (HBeAg) is a nonstructural protein produced during HBV replication and shares 90% amino acids with the nucleocapsid HBcAg; act to activate various regulated signal transduction pathways and, in the nucleus, regulate transcription by direct interaction with various transcription factors and, in some cases, enhance their binding to specific transcriptional elements. It is a non-structural protein (HBx) that functions.

HBV is one of the few DNA viruses that utilizes reverse transcriptase in a replication process that involves multiple steps, including entry, uncoating, and export of the viral genome to the nucleus. Initially, replication of the HBV genome involves the generation of RNA intermediates, which are then reverse transcribed to create the DNA viral genome.

When a cell is infected with HBV, the viral genome relaxed circular DNA (rcDNA) is transported to the cell nucleus and converted to episomal covalently closed circular DNA (cccDNA), which serves as a transcription template for viral mRNA. After transcription and nuclear export, cytoplasmic viral pregenomic RNA (pgRNA) assembles with HBV polymerase and capsid proteins to form the nucleocapsid, within which polymerase-catalyzed reverse transcription generates negative-strand DNA, which is subsequently converted into positive-strand DNA. It is copied into stranded DNA to form the progeny rcDNA genome. The mature nucleocapsid is then packaged into viral envelope proteins and emerges as a virion particle, or shuttled to the nucleus and amplifies the cccDNA reservoir via the intracellular cccDNA amplification pathway. cccDNA is an essential component of the HBV replication cycle and is involved in the establishment of infection and viral persistence.

HBV infection results in the production of two distinct particles: 1) the HBV virus itself (or Dane particle), which contains a viral capsid built from HBcAg and is coated by HBsAg, capable of reinfecting cells; and 2) subviral particles (or SVPs), which are high-density lipoprotein-like particles composed of lipids, cholesterol, cholesterol esters, and non-infectious small and medium forms of the hepatitis B surface antigen HBsAg. be. For each virus particle produced, 1,000-10,000 SVPs are released into the blood. SVPs (and the HBsAg proteins they carry) thus represent the overwhelming majority of viral proteins in the blood. HBV-infected cells also secrete a soluble proteolytic product of precore protein called HBVe antigen (HBeAg).

Eight genotypes of HBV, designated AH, have been determined, each with a different geographic distribution. The virus is non-cytopathic and virus-specific cellular immunity is responsible for the outcome of exposure to HBV - acute infection with resolution of liver disease in 6 months, or chronic HBV infection with frequent progressive liver injury. - is the main factor determining

The term "HBV" includes any of these eight HBV genotypes (A-H). The amino acid and complete coding sequences of the reference sequences of the HBV genome can be found, for example, in GenBank accession numbers GI:21326584 (SEQ ID NO: 1) and GI:3582357 (SEQ ID NO: 3).

Additional examples of HBV mRNA sequences are readily available using publicly available databases such as GenBank, UniProt, and OMIM.

The term "HBV" as used herein also refers to naturally occurring DNA sequence variants of the HBV genome.

As used herein, the term "hepatitis D virus" used synonymously with the term "HDV" refers to a well-known non-cytopathic hepatotropic DNA virus belonging to the Hepadnaviridae family. . For example, Ciancio and Rizzetto, Nat. Rev. 11:68-71, 2014; Le Gal et al. , Emerg. Infect. Dis. 12:1447-1450, 2006; and Abbas and afzal, World J. Hep. , 5:666-675, 2013 (all of which are incorporated by reference). Unless otherwise indicated, HDV refers to all clades and variants of HDV.

HDV produces one protein, HDAg. It has two forms, the 27 kDa large HDAg (also referred to herein as lHD, L-HDAg, and large HDV antigen) and the 24 kDa small HDAg (also referred to herein as sHD, S-HDAg, and small (also called HDV antigen). The N-terminus of these two forms is the same, and the C-terminus of the large HDAg differs by 19 amino acids. Both isoforms are produced from the same reading frame containing a UAG stop codon at codon 196 and usually produce only small HDAg. However, editing by the cellular enzyme adenosine deaminase-1 changes the stop codon to UCG, allowing the production of large HDAg. Despite having 90% sequence identity, these two proteins play different roles in the infection process. HDAg-S is produced early in infection, enters the nucleus, and assists virus replication. In contrast, HDAg-L is produced late in infection, acts as an inhibitor of viral replication, and is required for viral particle assembly.

Additional examples of HDV mRNA sequences are readily available using publicly available databases such as GenBank, UniProt, and OMIM.

The term "HDV" as used herein also refers to naturally occurring DNA sequence variants of the HDV genome.

As used herein, "target sequence" refers to a contiguous portion of the nucleotide sequence of an mRNA molecule formed during transcription of the HBV gene, including the mRNA that is the product of RNA processing of the primary transcript. . In one embodiment, the targeting portion of the sequence at least serves as a substrate for iRNA-directed cleavage at or near that portion of the nucleotide sequence of the mRNA molecule formed upon transcription of the HBV gene. It should be long enough.

The target sequence can be about 9-36 nucleotides in length, such as about 15-30 nucleotides in length. For example, the target sequence may include about 15-30 nucleotides, 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15- 20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19- 20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, It can be 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 nucleotides long. Ranges and lengths intermediate to those recited above are also considered to be part of the invention.

As used herein, the term "strand comprising a sequence" refers to an oligonucleotide comprising a strand of nucleotides represented by the sequence set forth using standard nucleotide nomenclature.

"G", "C", "A" and "U" each generally represent nucleotides containing guanine, cytosine, adenine, and uracil as bases, respectively. However, it will be appreciated that the term "ribonucleotide" or "nucleotide" may also refer to a modified nucleotide, or a surrogate replacement moiety, as detailed further below (see e.g. Table 2). ). Those skilled in the art will appreciate that guanine, cytosine, adenine, and uracil may be substituted by other moieties without appreciably altering the base pairing properties of oligonucleotides containing nucleotides with such substituted moieties. It has recognized. For example, without limitation, a nucleotide containing inosine as its base may base pair with a nucleotide containing adenine, cytosine, or uracil. Thus, nucleotides containing uracil, guanine, or adenine may be replaced, for example, by nucleotides containing inosine in the nucleotide sequences of the dsRNAs featured in the invention. In another example, adenine and cytosine anywhere in the oligonucleotide can be replaced with guanine and uracil, respectively, to form a GU wobble base pairing with the target mRNA. Sequences containing such substituted moieties are suitable for the compositions and methods featured in this invention.

The terms "iRNA," "RNAi agent," "iRNA agent," and "RNA interfering agent" used interchangeably herein refer to an RNA-containing agent, as that term is defined herein. , and refers to agents that mediate targeted cleavage of RNA transcripts through the RNA-induced silencing complex (RISC) pathway. iRNA directs sequence-specific degradation of mRNA by a process known as RNA interference (RNAi). The iRNA modulates (eg, inhibits) the expression of an HBV gene (eg, one or more HBV genes) within a cell, eg, a cell in a subject, such as a mammalian subject.

In one embodiment, the RNAi agent of the invention comprises a single-stranded RNA that interacts with a target RNA sequence, eg, an HBV target mRNA sequence, leading to cleavage of the target RNA. Without wishing to be bound by theory, it is believed that long double-stranded RNAs introduced into cells are degraded into siRNAs by a type III endonuclease known as Dicer (Sharp et al. (2001) ) Genes Dev. 15:485). Dicer, a ribonuclease III-like enzyme, processes dsRNA into short interfering RNAs of 19-23 base pairs with a characteristic two base 3' overhang (Bernstein, et al., (2001) Nature 409 :363). The siRNA is then incorporated into the RNA-induced silencing complex (RISC), where one or more helicases unwind the siRNA duplex and the complementary antisense strand guides target recognition. (Nykanen, et al., (2001) Cell 107:309). Upon binding to the appropriate target mRNA, one or more endonucleases in RISC cleave the target and induce silencing (Elbashir, et al., (2001) Genes Dev. 15:188). Here, in one aspect, the invention relates to single-stranded siRNA (ssRNA) that is produced in a cell and promotes the formation of a RISC complex that results in the silencing of a target gene, ie, an HBV gene. Accordingly, the term "siRNA" is also used herein to refer to RNAi as described above.

In another embodiment, the RNAi agent can be a single-stranded siRNA that is introduced into a cell or organism to inhibit a target mRNA. The single-stranded RNAi agent binds to the RISC endonuclease Argonaute 2, which in turn cleaves the target mRNA. Single-stranded siRNAs are generally 15-30 nucleotides and are chemically modified. Single-stranded siRNA design and testing is described in U.S. Pat. No. 8,101,348 and Lima et al. , (2012) Cell 150:883-894, each of which is incorporated herein by reference in its entirety. Any of the antisense nucleotide sequences described herein may be as described herein or as described by Lima et al. , (2012) Cell 150;: 883-894.

In another embodiment, "iRNA" for use in the compositions, uses and methods of the invention is double-stranded RNA, herein referred to as "double-stranded RNAi agent", "double-stranded RNA ( dsRNA) molecule,” “dsRNA agent,” or “dsRNA.” The term "dsRNA" refers to a target RNA, i.e., two antiparallel and substantially complementary nucleic acid strands that are shown to have a "sense" and "antisense" orientation with respect to the HBV gene. Refers to a complex of ribonucleic acid molecules that has a chain structure. In certain embodiments of the invention, double-stranded RNA (dsRNA) causes degradation of target RNA, eg, mRNA, by a post-transcriptional gene silencing mechanism, referred to herein as RNA interference or RNAi.

Generally, the majority of the nucleotides on each strand of a dsRNA molecule are ribonucleotides, but as described in detail herein, each or both strands may include one or more non-ribonucleotides, For example, deoxyribonucleotides and/or modified nucleotides may also be included. Additionally, as used herein, an "RNAi agent" may include ribonucleotides with chemical modifications; an RNAi agent may include substantial modifications at multiple nucleotides.
As used herein, the term "modified nucleotide" refers to a nucleotide that independently has a modified sugar moiety, a modified internucleotide linkage, and/or a modified nucleobase. Thus, the term modified nucleotide encompasses, for example, substitutions, additions, or removals of functional groups or atoms to internucleoside linkages, sugar moieties, or nucleobases. Modifications suitable for use in the agents of the invention include any type of modification disclosed herein or known in the art. Any such modification when used on siRNA type molecules is encompassed by "RNAi agent" for purposes of this specification and claims.

The double-stranded region may be of any length that allows specific degradation of the desired target RNA via the RISC pathway, and may be about 9 to 36 base pairs in length, such as about 15 ~30 base pairs in length, e.g., about 15-30, 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15 ～21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23 , 18-22, 18-21, 18-20, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19 ～21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30 , 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 base pairs in length, about 9, 10, 11, 12, 13 , 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 base pairs It can be any length. Ranges and lengths intermediate to those recited above are also considered to be part of the invention.

The two strands forming the double-stranded structure may be different parts of one larger RNA molecule, or they may be separate RNA molecules. A sequence of nucleotides between the 3' end of one strand and the 5' end of the other strand where the two strands are part of one larger molecule, thus forming a double-stranded structure When joined by strands that are connected together, the joining RNA strands are called "hairpin loops." A hairpin loop can include at least one unpaired nucleotide. In certain embodiments, the hairpin loops are at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 20, at least It may contain 23 or more unpaired nucleotides.

When the two substantially complementary strands of dsRNA are constituted by separate RNA molecules, the molecules may or may not be covalently linked. A bond when two strands are covalently linked by other means than a continuous strand of nucleotides between the 3' end of one strand and the 5' end of the other strand forming a double-stranded structure. The structure is called a "linker." RNA strands can have the same or different number of nucleotides. The maximum number of base pairs is the number of nucleotides in the shortest strand of the dsRNA minus any overhang present in the duplex. In addition to a double-stranded structure, an RNAi agent may contain one or more nucleotide overhangs.

In one embodiment, the RNAi agent of the invention is a dsRNA, each strand of which comprises 24-30 nucleotides that interact with a target RNA sequence, eg, an HBV target mRNA sequence, leading to cleavage of the target RNA. Without wishing to be bound by theory, long double-stranded RNA introduced into cells is degraded into siRNA by a type III endonuclease known as Dicer (Sharp et al. (2001) Genes Dev. 15:485). Processing of dsRNA by the ribonuclease-III-like enzyme Dicer results in small interfering RNAs of 19-23 base pairs with a characteristic 2 base 3' overhang (Bernstein, et al., (2001) Nature 409:363). The siRNA is then integrated into the RNA-induced silencing complex (RISC), where one or more helicases unwind the siRNA duplex, allowing the complementary antisense strand to guide target recognition (Nykanen , et al., (2001) Cell 107:309). Upon binding to the appropriate target mRNA, one or more endonucleases within RISC cleave the target and induce silencing (Elbashir, et al., (2001) Genes Dev. 15:188).

As used herein, the term "nucleotide overhang" refers to at least one unpaired nucleotide that protrudes from the double-stranded structure of an iRNA, such as a dsRNA. For example, a nucleotide overhang exists when the 3' end of one strand of the dsRNA extends beyond the 5' end of the other strand, or vice versa. The dsRNA can include an overhang of at least one nucleotide; or the overhang can include at least 2 nucleotides, at least 3 nucleotides, at least 4 nucleotides, at least 5 nucleotides, or more. Nucleotide overhangs may include or consist of nucleotide/nucleoside analogs, including deoxynucleotides/nucleosides. The overhang can be the sense strand, antisense strand or any combination thereof. Additionally, overhanging nucleotides can be present at the 5' end, 3' end, or both ends of either the antisense or sense strand of the dsRNA.

In one embodiment, the antisense strand of the dsRNA has 1 to 10 nucleotides at the 3' and/or 5' end, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or with an overhang of 10 nucleotides. In one embodiment, the sense strand of the dsRNA has 1 to 10 nucleotides at the 3' end and/or 5' end, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 has an overhang of nucleotides. In another embodiment, one or more of the nucleotides in the overhang are replaced with a nucleoside thiophosphate.

"Blunt" or "blunt end" means that there are no unpaired nucleotides at the relevant end of the double-stranded RNAi agent, ie, there are no nucleotide overhangs. A "blunt-ended" RNAi agent is a dsRNA that is double-stranded over its entire length, ie, there are no nucleotide overhangs at either end of the molecule. RNAi agents of the invention include RNAi agents that have a nucleotide overhang at one end (ie, agents that have one overhang and one blunt end) or RNAi agents that have nucleotide overhangs at both ends.

The term "antisense strand" or "guide strand" refers to a strand of an iRNA, eg, dsRNA, that includes a region that is substantially complementary to, eg, a target sequence, eg, HBV mRNA. As used herein, the term "region of complementarity" refers to a sequence, e.g., an antisense strand that is substantially complementary to a target sequence, e.g., an HBV nucleotide sequence, as defined herein. refers to the area of If the regions of complementarity are not completely complementary to the target sequence, there may be mismatches in internal or terminal regions of the molecule. Generally, most tolerated mismatches are in the terminal regions, eg, 5, 4, 3, 2, or 1 nucleotide at the 5' and/or 3' ends of the iRNA. In one embodiment, a double-stranded RNAi agent of the invention contains a nucleotide mismatch in the antisense strand. In another embodiment, a double-stranded RNAi agent of the invention contains a nucleotide mismatch in the sense strand. In one embodiment, the nucleotide mismatch is, for example, within 5, 4, 3, 2, or 1 nucleotide from the 3' end of the iRNA. In another embodiment, the nucleotide mismatch is, for example, at the 3' terminal nucleotide of the iRNA.

The term "sense strand" or "passenger strand" as used herein refers to a region that is substantially complementary to a region of the antisense strand, as that term is defined herein. Refers to the strand of iRNA containing

As used herein, the term "cleavage region" refers to the region located immediately adjacent to the cleavage site. The cleavage site is the site in the target where cleavage occurs. In certain embodiments, the cleavage region includes three bases directly adjacent to the cleavage site at either end of the cleavage site. In certain embodiments, the cleavage region includes two bases immediately adjacent to the cleavage site at either end of the cleavage site. In certain embodiments, the cleavage site occurs specifically at the site bound by nucleotides 10 and 11 of the antisense strand, and the cleavage region includes nucleotides 11, 12, and 13.

As used herein, unless otherwise indicated, the term "complementary" refers to describing a first nucleotide sequence in relation to a second nucleotide sequence, as understood by those skilled in the art. When used in a method, an oligonucleotide or polynucleotide comprising a first nucleotide sequence hybridizes under predetermined conditions with an oligonucleotide or polynucleotide comprising a second nucleotide sequence to form a double-stranded structure. Refers to ability. Such conditions may be, for example, stringent conditions, where stringent conditions include 400 mM NaCl, 40 mM PIPES (pH 6.4), 1 mM EDTA at 50°C or 70°C for 12 to 16 hours followed by washing (see, e.g., “Molecular Cloning: A Laboratory Manual, Sambrook, et al. (1989) Cold Spring Harbor Laboratory Press). conditions etc. Other conditions can be applied.A person skilled in the art will be able to determine the most appropriate set of conditions for testing the complementarity of two sequences, according to the ultimate use of the hybridized nucleotides. Dew.

Complementary sequences in the iRNAs described herein, e.g., in dsRNAs, may be one of the oligonucleotides or polynucleotides comprising a first nucleotide sequence to an oligonucleotide or polynucleotide comprising a second nucleotide sequence. or comprises base pairing over the entire length of both nucleotide sequences. Such sequences may be referred to herein as "fully complementary" to each other. However, when a first sequence is referred to herein as "substantially complementary" to a second sequence, the two sequences may be completely complementary, or their final When hybridizing to duplexes of up to 30 base pairs, they One or more, but generally no more than 5, no more than 4, no more than 3, or no more than 2 mismatched base pairs may be formed. However, if two oligonucleotides are designed to form one or more single-stranded overhangs after hybridization, such overhangs shall not be considered a mismatch for purposes of determining complementarity. do. For example, for purposes described herein, a dsRNA comprising one oligonucleotide that is 21 nucleotides long and another oligonucleotide that is 23 nucleotides long is defined as a dsRNA in which the longer oligonucleotide overlaps the shorter oligonucleotide. may be referred to as "fully complementary" if it contains a sequence of 21 nucleotides that is completely complementary to.

"Complementary" sequences as used herein are formed from non-Watson-Crick base pairs and/or non-natural and modified nucleotides, so long as the above requirements related to their ability to hybridize are met. base pairs, or may be formed entirely from such base pairs. Such non-Watson-Crick base pairs include, but are not limited to, G:U wobble or Hoogsteen type base pairs.

As used herein, the terms "complementary," "fully complementary," and "substantially complementary" refer to the sense and antisense strands of a dsRNA, as understood from the context of their use. or between the antisense strand of the dsRNA and the target sequence.

As used herein, a polynucleotide "substantially complementary to at least a portion of" messenger RNA (mRNA) includes the 5'UTR, open reading frame (ORF), or 3'UTR of interest. Refers to a polynucleotide that is substantially complementary to a contiguous portion of mRNA (eg, mRNA encoding an HBV gene). For example, a polynucleotide is complementary to at least a portion of an HBV mRNA if its sequence is substantially complementary to a contiguous portion of the mRNA encoding the HBV gene.

Thus, in some embodiments, the antisense strand polynucleotides disclosed herein are fully complementary to the target HBV sequence. In other embodiments, the antisense strand polynucleotides disclosed herein are substantially complementary to a target HBV sequence and include the nucleotide sequence of SEQ ID NO: 1, or an equivalent region of a fragment of SEQ ID NO: 1. at least about 80% complementary over its entire length, such as about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94% %, about 95%, about 96%, about 97%, about 98%, or about 99% complementary contiguous nucleotide sequences.

In one embodiment, an RNAi agent of the invention comprises a sense strand substantially complementary to an antisense polynucleotide, the antisense polynucleotide in turn being complementary to a target HBV sequence, and wherein the sense strand polynucleotide is complementary to a target HBV sequence. The nucleotide is a nucleotide sequence of any one of SEQ ID NOs: 6, 8, 10, 12, 38, and 40, or an equivalent region of a fragment of any one of SEQ ID NOs: 6, 8, 10, 12, 38, and 40. and at least about 80% complementary over its entire length, such as about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, Contains contiguous nucleotide sequences that are about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% complementary. In another embodiment, the RNAi agent of the invention comprises an antisense strand substantially complementary to the target HBV sequence, the nucleotide sequence of any one of SEQ ID NOs: 5, 7, 9, 11, 37, and 39; or at least about 80% complementary over its entire length to the equivalent region of any one of the fragments of SEQ ID NOs: 5, 7, 9, 11, 37, and 39, such as about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% complementary Contains a contiguous nucleotide sequence.

In some embodiments, generally the majority of the nucleotides in each strand are ribonucleotides, but as described in detail herein, each or both strands may contain one or more ribonucleotides. Non-ribonucleotides such as deoxyribonucleotides and/or modified nucleotides may also be included. Additionally, "iRNA" can include ribonucleotides with chemical modifications. Such modifications may include any type of modification disclosed herein or known in the art. Any such modification when used on an iRNA molecule is encompassed by "iRNA" for purposes of this specification and claims.

In one aspect of the invention, agents for use in the methods and compositions of the invention are single-stranded antisense nucleic acid molecules that inhibit target mRNA via an antisense inhibition mechanism. Single-stranded antisense RNA molecules are complementary to sequences in the target mRNA. Single-stranded antisense oligonucleotides can stoichiometrically inhibit translation by base-pairing to mRNA and physically interfering with the translation machinery (Dias, N. et al., (2002) Mol Cancer Ther 1:347-355). Single-stranded antisense RNA molecules are about 15 to about 30 nucleotides in length and can have a sequence that is complementary to a target sequence. For example, a single-stranded antisense RNA molecule comprises at least about 15, 16, 17, 18, 19, 20, or more contiguous nucleotides from any one of the antisense sequences described herein. may contain an array that is .

As used herein, "subject" refers to primates (humans, non-human primates such as monkeys, chimpanzees, etc.), non-primates (cows, pigs, camels, llamas, horses, goats, etc.) Animals such as mammals, including rabbits, sheep, hamsters, guinea pigs, cats, dogs, rats, mice, horses, and whales), or birds (eg, ducks or geese). In certain embodiments, the subject is a human being treated or being evaluated for a disease, disorder, or condition that could benefit from reducing HBV gene expression and/or replication; as described herein; A person at risk for a disease, disorder or condition that could benefit from reducing HBV gene expression and/or replication; and/or a person at risk for a disease, disorder or condition that could benefit from reducing HBV gene expression and/or replication. A human being, such as a human being being treated for a disease, disorder or condition that could benefit from a reduction in gene expression and/or replication. In another embodiment, the subject has a hepatitis B virus (HBV) infection. In another embodiment, the subject has both a hepatitis B virus (HBV) infection and a hepatitis D virus (HDV) infection.

As used herein, the term "treat" or "treatment" refers to, but is not limited to, treating one or more conditions associated with undesirable HBV gene expression and/or HBV replication, such as serum and/or Presence of hepatic HBV ccc DNA, presence of serum and/or liver HBV antigens such as HBsAg and/or HBeAg, elevated ALT, elevated AST, absence or low levels of anti-HBV antibodies, liver injury; cirrhosis; delta hepatitis, B acute hepatitis B; acute fulminant hepatitis B; chronic hepatitis B; liver fibrosis; end-stage liver disease; hepatocellular carcinoma; serum sickness-like syndrome; anorexia; nausea; vomiting, mild fever; myalgia; easy fatigue loss of taste and smell (food and tobacco aversion); and/or right upper quadrant and epigastric pain (intermittent, mild to moderate); hepatic encephalopathy; somnolence; disturbed sleep patterns; mental confusion; coma; ascites ; Gastrointestinal bleeding; coagulopathy; jaundice; hepatomegaly (slightly enlarged and soft liver); splenomegaly; palmar erythema; spider nevus; muscle wasting; spider hemangioma; vasculitis; variceal hemorrhage; peripheral edema; gynecomastia; testicular atrophy; abdominal collateral veins (caput medusa); high levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST), in the range of 1000-2000 IU/mL; However, values 100 times higher than the upper limit of normal range (ULN) may also be identified; ALT levels higher than AST levels; elevated γ-glutamyl transpeptidase (GGT) and alkaline phosphatase (ALP) levels (e.g., 3 times higher than ULN) ); moderately low albumin levels; elevated serum iron levels; leukopenia (i.e., granulocytopenia); lymphocytosis; increased erythrocyte sedimentation rate (ESR); shortened red blood cell lifespan; hemolysis; thrombocytopenia; Prolongation of international normalized ratio (INR); presence of serum and/or liver HBsAg, HBeAg, hepatitis B core antibodies (anti-HBc) immunoglobulin M (IgM); hepatitis B surface antibodies (anti-HBs), hepatitis B e Antibodies (anti-HBe) and/or HBV DNA; elevated aminotransferases (≤5 times ULN); ALT levels higher than AST levels; increased bilirubin levels, prolongation of prothrombin time (PT); hyperglobulinemia; Presence of tissue non-specific antibodies such as smooth muscle antibodies (ASMA) or antinuclear antibodies (ANA) (10-20%); presence of tissue-specific antibodies such as antibodies to the thyroid (10-20%); rheumatoid factor ( RF); hyperbilirubinemia, prolonged PT, decreased platelet and white blood cell counts, AST levels higher than ALT levels; increased levels of alkaline phosphatase (ALP) and GGT; lobular, degenerating and reparative hepatocytes. Refers to a beneficial or desired outcome, including the reduction or amelioration of predominantly centrilobular necrosis, whether detectable or undetectable; and associated inflammation; "Treatment" can also mean prolonging survival as compared to expected survival in the absence of treatment.

The term "lower" in the context of a subject's level of HBV gene expression and/or HBV replication or disease marker or symptom refers to a statistically significant reduction in such level. The reduction may be, for example, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least It may be 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or more, preferably recognized as within the normal range for individuals without such disorder. level. In certain embodiments, the expression of the target is normalized, i.e., reduced to a level that is accepted as within the normal range for individuals not having such disorder, e.g., the level of a disease marker such as ALT or AST is reduced to a level that is accepted as within the normal range for individuals without the disorder.

As used herein, "prophylaxis" or "preventing" when used in reference to a disease, disorder or condition thereof that may benefit from reduction of HBV gene expression and/or replication; symptoms associated with such a disease, disorder, or condition in the subject, such as the presence of serum and/or liver HBV ccc DNA, the presence of serum HBV DNA, the presence of serum and/or liver HBV antigens, such as HBsAg and/or HBeAg; Increased ALT, increased AST, absence or low levels of anti-HBV antibodies, liver injury; cirrhosis; delta hepatitis, acute hepatitis B; acute fulminant hepatitis B; chronic hepatitis B; liver fibrosis; end-stage liver disease; Hepatocellular carcinoma; serum sickness-like syndrome; anorexia; nausea; vomiting, mild fever; myalgia; fatigue; impaired taste and smell (food and tobacco aversion); and/or right upper quadrant pain and epigastric pain ( hepatic encephalopathy; somnolence; disturbed sleep patterns; mental confusion; coma; ascites; gastrointestinal bleeding; coagulopathy; jaundice; hepatomegaly (mildly enlarged and soft liver); splenomegaly; palmar Erythema; spider nevus; muscle wasting; spider hemangioma; vasculitis; variceal hemorrhage; peripheral edema; gynecomastia; testicular atrophy; abdominal collateral veins (caput medusa); Levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) within the range of 1000-2000 IU/mL, but 100 times higher than the upper limit of normal range (ULN) may also be identified; ALT levels higher than AST levels elevated γ-glutamyl transpeptidase (GGT) and alkaline phosphatase (ALP) levels (e.g., less than 3 times ULN); moderately low albumin levels; elevated serum iron levels; leukopenia (i.e., granulocytopenia) ; lymphocytosis; increased erythrocyte sedimentation rate (ESR); shortened red blood cell lifespan; hemolysis; thrombocytopenia; prolonged international normalized ratio (INR); serum and/or liver HBsAg, HBeAg, hepatitis B core antibodies ( presence of immunoglobulin M (IgM); hepatitis B surface antibodies (anti-HBs), hepatitis B e antibodies (anti-HBe), and/or HBV DNA; elevated aminotransferases (≤5 times ULN); ALT levels higher than AST levels; increased bilirubin levels, prolonged prothrombin time (PT); hyperglobulinemia; presence of tissue nonspecific antibodies such as anti-smooth muscle antibodies (ASMA) or anti-nuclear antibodies (ANA) (10 ~20%); presence of tissue-specific antibodies (10-20%), such as antibodies against the thyroid; increased levels of rheumatoid factor (RF); hyperbilirubinemia, prolonged PT, decreased platelet and white blood cell counts, ALT AST levels higher than normal levels; elevated alkaline phosphatase (ALP) and GGT levels; lobular, degenerative and reparative hepatocellular changes, and concomitant inflammation; detectable or undetectable, predominantly centrilobular Refers to a reduction in the likelihood of developing undesirable symptoms of HBV infection such as necrosis. For example, the likelihood of developing liver fibrosis may vary depending on whether an individual with one or more risk factors for liver fibrosis, e.g., chronic hepatitis B infection, does not develop liver fibrosis, or an individual with the same risk factors and the present It is reduced when liver fibrosis develops at a lower severity compared to the population not receiving treatment as described. the absence of the disease, disorder or condition, or a reduction in the occurrence of symptoms associated with such disease, disorder or condition (e.g., by at least about 10% on a clinically accepted scale for the disease or disorder); , or a delay in symptoms (eg, days, weeks, months, or years) is considered effective prevention.

As used herein, the term "hepatitis B virus-associated disease" or "HBV-associated disease" is a disease or disorder caused by or associated with HBV infection and/or replication. The term "HBV-related disease" includes any disease, disorder or condition that could benefit from reduction of HBV gene expression and/or replication. Non-limiting examples of HBV-related diseases include, for example, hepatitis D virus infection, delta hepatitis, acute hepatitis B; acute fulminant hepatitis B; chronic hepatitis B; liver fibrosis; end-stage liver disease; Cancer is an example.

In one embodiment, the HBV-related disease is hepatitis D virus infection. Hepatitis D virus or hepatitis delta virus (HDV) is a human pathogen. However, this virus is a defective virus and transmission depends on essential helper functions provided by hepatitis B virus (HBV); in fact, for HDV to become infectious and actively multiply, it is necessary to HBV infection, specifically a viral envelope containing the surface antigen of hepatitis B, is required. HDV can lead to severe acute and chronic liver disease associated with HBV. Hepatitis D infection and/or hepatitis delta is highly endemic in some African countries, the Amazon region, and the Middle East, while its prevalence is low in industrialized countries, except in the Mediterranean region.

Transmission of HDV can occur through co-infection with HBV (co-infection) or can occur superimposed on chronic hepatitis B or hepatitis B carriage (superinfection). Both co-infection and co-infection with HDV result in more severe complications compared to infection with HBV alone. These complications include an increased likelihood of developing liver failure in acute infections, and rapid progression of cirrhosis and increased likelihood of developing liver cancer in chronic infections. In combination with hepatitis B virus, hepatitis D has the highest mortality rate of all hepatitis infections at 20%.

In one embodiment, the HBV-related disease is acute hepatitis B. Acute hepatitis B involves inflammation of the liver that lasts less than six months. Typical symptoms of acute hepatitis B are fatigue, anorexia, nausea, and vomiting. Very high aminotransferase values (>1000 U/L) and hyperbilirubinemia are often observed. Severe cases of acute hepatitis B can rapidly progress to acute liver failure, which is characterized by synthetic dysfunction of the liver. This is often defined as a prothrombin time (PT) of 16 seconds or an international normalized ratio (INR) of 1.5 in the absence of prior liver disease. Acute hepatitis B can progress to chronic hepatitis B.

In one embodiment, the HBV-related disease is chronic hepatitis. Chronic hepatitis B (CHB) involves inflammation of the liver that lasts for more than six months. Subjects with chronic hepatitis B disease exhibit immune tolerance or have an inactive chronic infection with no evidence of active disease, and these subjects are also asymptomatic. Patients with chronic active hepatitis may have symptoms similar to acute hepatitis, especially during the replicative state. Persistence of HBV infection in CHB subjects is due to ccc HBV DNA. In one embodiment, the subject with CHB is HBeAg positive. In another embodiment, the subject with CHB is HBeAg negative. Subjects with CHB have serum HBV DNA levels of less than about 105 and persistent elevations in transaminases such as ALT, AST and γ-glutamyltransferase. A subject with CHB may have a liver biopsy score (eg, necroinflammatory score) of less than about 4. Additionally, subjects with CHB may have.

In one embodiment, the HBV-related disease is acute fulminant hepatitis B. Subjects with acute fulminant hepatitis B experience symptoms of acute hepatitis, plus confusion or coma (due to the inability of the liver to detoxify chemicals) and purpura or hemorrhage (due to lack of blood clotting factors). Have symptoms.

Subjects with HBV infection, such as CHB, can develop liver fibrosis. Thus, in one embodiment, the HBV-related disease is liver fibrosis. Liver fibrosis, or cirrhosis, is histologically defined as diffuse liver lesions characterized by fibrosis (excess fibrous connective tissue) and changes from normal liver structure to structurally abnormal nodules. Ru.

Subjects with HBV infection, such as CHB, can develop end-stage liver disease. Thus, in one embodiment, the HBV-related disease is end-stage liver disease. For example, liver fibrosis can progress to the point where the body can no longer compensate for, for example, decreased liver function as a result of liver fibrosis, causing, for example, mental and neurological symptoms and liver failure.

Subjects with HBV infection, such as CHB, can develop hepatocellular carcinoma (HCC), also referred to as malignant liver cancer. Thus, in one embodiment, the HBV-related disease is HCC. HCC often develops in subjects with CHB and can be fibrolamellar, pseudoductal (adenoidoid), pleomorphic (giant cell), or clear cell.

An "HDV-related disorder" or "hepatitis D virus-related disorder" is a disease or disorder associated with the expression of HDV. Exemplary HDV-related disorders include hepatitis B virus infection, acute hepatitis B, acute hepatitis D; acute fulminant hepatitis D; chronic hepatitis D; liver fibrosis; end-stage liver disease; and Examples include hepatocellular carcinoma.

A "therapeutically effective amount," as used herein, when administered to a patient to treat a subject with an HBV infection and/or an HBV-related disease, is effective in treating a disease (e.g. It is intended to include an amount of RNAi agent sufficient to cause (by attenuating, ameliorating, or maintaining one or more symptoms). A "therapeutically effective amount" refers to the RNAi agent, the method of administration of the agent, the disease and its severity and the stage of the pathological process mediated by medical history, age, weight, family history, genetic makeup, HBV gene expression, and the stage of the pathological process present. It may vary depending on the type of prior or concomitant treatment and other individual characteristics of the patient being treated.

A "prophylactically effective amount," as used herein, refers to a disease or It is intended to include an amount of RNAi agent sufficient to prevent or ameliorate one or more symptoms of the disease. Ameliorating a disease includes slowing the course of the disease or reducing the severity of a disease that later develops. A "preventive effective amount" is determined based on the RNAi agent, the method of administering the agent, the degree of disease risk, and medical history, age, weight, family history, genetic makeup, type of prior or concomitant treatment, if any, and the intended treatment. and may vary depending on other individual characteristics of the patient.

A "therapeutically effective amount" or "prophylactically effective amount" also includes an amount of an RNAi agent that produces any desired local or systemic effect at a reasonable benefit/risk ratio applicable to any treatment. . The RNAi agents used in the methods of the invention can be administered in amounts sufficient to yield a reasonable benefit/risk ratio applicable to such treatments.

The term "sample" as used herein includes similar bodily fluids, cells, or tissues taken from a subject, as well as collections of bodily fluids, cells, or tissues present within the subject's body. Examples of biological fluids include blood, serum and serous fluids, plasma, cerebrospinal fluid, eye fluid, lymph fluid, urine, saliva, and the like. Tissue samples can include samples from tissues, organs or localized areas. For example, the sample may be derived from a particular organ, part of an organ, or body fluids or cells within those organs. In certain embodiments, the sample is a liver (e.g., whole liver or a particular portion of the liver or a particular type of cell within the liver, such as, for example, hepatocytes), a retina, or a portion of the retina (e.g., retinal pigment epithelium). , the central nervous system or a part of the central nervous system (eg, the ventricles or choroid plexus), or the pancreas or a particular cell or part of the pancreas. In some embodiments, a "sample derived from a subject" refers to cerebrospinal fluid obtained from a subject. In a preferred embodiment, a "sample derived from a subject" refers to blood or plasma collected from a subject. In a further embodiment, a "sample derived from a subject" refers to liver tissue (or a partial component thereof) or retinal tissue (or a partial component thereof) derived from a subject.

II. iRNA of the present invention
The invention provides iRNAs that inhibit the expression of one or more HBV genes. In one embodiment, the iRNA agent is administered within a cell, such as a cell within a subject, e.g., a mammal, such as a human, suffering from a disease or disorder associated with HBV, including but not limited to chronic hepatitis B. Contains double-stranded ribonucleic acid (dsRNA) molecules for inhibiting HBV gene expression. The dsRNA includes an antisense strand that has a region of complementarity that is complementary to at least a portion of the mRNA formed during expression of the HBV gene. The region of complementarity is about 30 nucleotides or less in length (eg, about 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, or 18 nucleotides or less in length). Upon contact with cells expressing HBV genes, iRNA can be isolated using e.g. PCR or branched DNA (bDNA) based methods, or protein-based e.g. by immunofluorescence analysis using Western blotting or flow cytometry techniques. inhibits HBV gene expression by at least about 10% as assayed by the method.

dsRNA includes two RNA strands that are complementary and hybridize to form a double-stranded structure under the conditions under which the dsRNA is used. One strand of the dsRNA (the antisense strand) contains a region of complementarity that is substantially complementary, generally completely complementary, to the target sequence. The target sequence may be derived from the sequence of the mRNA formed during expression of the HBV gene. The other strand (the sense strand) contains a region that is complementary to the antisense strand, allowing the two strands to hybridize and form a double-stranded structure when combined under suitable conditions. ing. As described elsewhere herein and as is known in the art, complementary sequences of dsRNA may also be present on one nucleic acid molecule rather than on separate oligonucleotides. It may also be included as a self-complementary region.

Generally, the double-stranded structure will have a length of 15-30 base pairs, e.g. , 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18 ~23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22 , 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21 ~30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 base pairs in length. Ranges and lengths intermediate to those recited above are also considered to be part of the invention.

Similarly, regions of complementarity of the target sequence may be 15-30 nucleotides long, e.g. 22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19- 22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 nucleotides in length. Ranges and lengths intermediate to those recited above are also considered to be part of the invention.

In certain embodiments, the dsRNA is about 15 to about 20 nucleotides in length, or about 25 to about 30 nucleotides in length. Generally, dsRNA is long enough to serve as a substrate for the Dicer enzyme. For example, it is well known in the art that dsRNAs longer than about 21-23 nucleotides in length can serve as substrates for Dicer. As those skilled in the art will also recognize, the region of RNA targeted for cleavage is most often part of a larger RNA molecule, often an mRNA molecule. In relevant cases, a "portion" of an mRNA target is a contiguous portion of the mRNA target of sufficient length to be a substrate for RNAi-directed cleavage (i.e., cleavage via the RISC pathway). It is an array.

The double-stranded region is the primary functional portion of the dsRNA, eg, about 9-36 base pairs, eg, about 10-36, 11-36, 12-36, 13-36, 14-36, 15-36, 9- 35, 10-35, 11-35, 12-35, 13-35, 14-35, 15-35, 9-34, 10-34, 11-34, 12-34, 13-34, 14-34, 15-34, 9-33, 10-33, 11-33, 12-33, 13-33, 14-33, 15-33, 9-32, 10-32, 11-32, 12-32, 13- 32, 14-32, 15-32, 9-31, 10-31, 11-31, 12-31, 13-32, 14-31, 15-31, 15-30, 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18- 29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20- 26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, Those skilled in the art will also recognize that it is a double-stranded region of 21-23, or 21-22 base pairs. Here, in one embodiment, RNA molecules or duplexes of more than 30 base pairs are targeted for cleavage to the extent that the desired RNA is processed into a functional duplex of, for example, 15-30 base pairs. A complex of RNA molecules with regions is dsRNA. Accordingly, those skilled in the art will recognize that in one embodiment, the miRNA is a dsRNA. In another embodiment, the dsRNA is not a naturally occurring miRNA. In another embodiment, iRNA agents useful for targeting expression of HBV genes are not generated in target cells by cleavage of larger dsRNAs.

The dsRNA described herein can further include one or more single-stranded nucleotide overhangs, eg, 1, 2, 3, or 4 nucleotides. dsRNAs with at least one nucleotide overhang may have unexpectedly superior inhibitory properties compared to their blunt-ended counterparts. Nucleotide overhangs may include or consist of nucleotide/nucleoside analogs, including deoxynucleotides/nucleosides. The overhang can be on the sense strand, antisense strand or any combination thereof. Additionally, overhanging nucleotides can be present on the 5' end, 3' end, or both ends of either the antisense or sense strand of the dsRNA.

dsRNA can be synthesized by standard methods known in the art, as described further below, for example, by the use of an automated DNA synthesizer (such as one commercially available from Biosearch, Applied Biosystems, Inc.). can be done.

iRNA compounds of the invention can be prepared using a two-step procedure. First, the individual strands of a double-stranded RNA molecule are prepared separately. Next, the component chains are annealed. Individual strands of siRNA compounds can be prepared using solution phase or solid phase organic synthesis or both. Organic synthesis offers the advantage that oligonucleotide chains containing non-natural or modified nucleotides can be easily prepared. Single-stranded oligonucleotides of the invention can be prepared using solution phase or solid phase organic synthesis or both.

In one embodiment, a dsRNA of the invention comprises at least two nucleotide sequences, a sense sequence and an antisense sequence. The sense strand is selected from the group of sequences shown in any one of Tables 3, 4, 6, 7, 12, 13, 22, 23, 25, and 26, and the corresponding antisense strand of the sense strand is: Selected from the group of sequences in any one of Tables 3, 4, 6, 7, 12, 13, 22, 23, 25, and 26. In this embodiment, one of the two sequences is complementary to the other of the two sequences, and one of the sequences is substantially complementary to the sequence of the mRNA produced during expression of the HBV gene. Therefore, in this embodiment, the dsRNA will include two oligonucleotides, where one oligonucleotide is one of the The second oligonucleotide is expressed as the sense strand in any one of Tables 3, 4, 6, 7, 12, 13, 22, 23, 25, and 26. Represented as the corresponding antisense strand. In one embodiment, the substantially complementary sequences of the dsRNA are contained in separate oligonucleotides. In another embodiment, the substantially complementary sequences of dsRNA are contained in one oligonucleotide.

Although some of the sequences in Tables 3, 4, 6, 7, 12, 13, 22, 23, 25, and 26 are described as modified and/or conjugated sequences, the RNA of the iRNA of the invention, e.g. dsRNAs of the invention are unmodified, unconjugated, and/or modified and/or conjugated differently than those listed in these tables, Tables 3, 4, 6, 7 , 12, 13, 22, 23, 25, and 26.

Those skilled in the art will appreciate that dsRNAs having a double-stranded structure of about 20-23 base pairs, such as 21 base pairs, have been found to be particularly effective in inducing RNA interference. (Elbashir et al., EMBO 2001, 20:6877-6888). However, others skilled in the art have found that shorter or longer RNA duplex structures may also be effective (Chu and Rana (2007) RNA 14:1714-1719; Kim et al. (2005) Nat Biotech 23:222-226). In the above embodiments, the dsRNA as described herein by the nature of the oligonucleotide sequence shown in any one of Tables 3, 4, 6, 7, 12, 13, 22, 23, 25, and 26. may include at least one strand of a minimum length of 21 nucleotides. shorter having one of the sequences in any one of Tables 3, 4, 6, 7, 12, 13, 22, 23, 25, and 26 minus a negligible number of nucleotides at one or both ends It can be reasonably expected that duplexes may be equally effective compared to the dsRNAs described above. Accordingly, at least 15, 16, 17, 18, 19, 20 from one of the sequences of any one of Tables 3, 4, 6, 7, 12, 13, 22, 23, 25, and 26, or more contiguous nucleotide sequence, the ability to inhibit HBV gene expression is about 5, 10, 15, 20, 25, or 30% less inhibition than the dsRNA containing the complete sequence. dsRNAs that differ by a.

Additionally, the RNAs shown in any one of Tables 3, 4, 6, 7, 12, 13, 22, 23, 25, and 26 identify sites in the HBV transcript that are susceptible to RISC-mediated cleavage. do. Therefore, the present invention further features iRNAs that target within one of these sites. As used herein, an iRNA is said to be targeted within a particular site of an RNA transcript if the iRNA promotes cleavage of the transcript anywhere within that particular site. Such iRNAs are generally linked to additional nucleotide sequences taken from regions flanking the selected sequences in the HBV gene. 25, and 26.

Target sequences are generally about 15-30 nucleotides in length, although the suitability of particular sequences within this range to direct cleavage of any given target RNA varies. The various software packages and guidelines described herein provide guidance for the identification of optimal target sequences for any given gene target, but for a given size (as a non-limiting example) , 21 nucleotides) literally or figuratively (including in silico) on the target RNA sequence to identify sequences within a size range that can serve as target sequences. ), you can also take an empirical approach. Next, by gradually moving the sequence "window" one nucleotide upstream or downstream of the initial target sequence position, until the complete set of possible sequences is identified for any given target size selected. potential target sequences can be identified. This process involves the systematic synthesis and testing of identified sequences (using assays described herein or known in the art) to identify sequences that function optimally. RNA sequences that mediate the best inhibition of target gene expression can be identified when targeted using a protein. Thus, for example, the sequences identified in any one of Tables 3, 4, 6, 7, 12, 13, 22, 23, 25, and 26 represent valid target sequences, but are comparable or more It is believed that further optimization of inhibition efficiency can be achieved by gradually "windowing" one nucleotide upstream or downstream of a given sequence to identify sequences with good inhibitory properties.

Further, for example, for any sequence specified in any one of Tables 3, 4, 6, 7, 12, 13, 22, 23, 25, and 26, Further optimization is achieved by testing the sequences generated by systematically adding or removing nucleotides to and moving longer or shorter size windows from that point up or down the target RNA. It is thought that it can be done. We also couple this approach for generating novel candidate targets with testing the efficacy of iRNAs based on their target sequences in inhibition assays known in the art and/or described herein. In this way, the efficiency of inhibition can be further improved. Furthermore, such optimized sequences can be modified, for example, by introducing modified nucleotides, adding or changing overhangs, as described herein or known in the art, or by further modifying the molecule as an expression inhibitor. Other modifications known in the art and/or described herein to optimize (e.g., increase serum stability or circulation half-life, increase thermal stability, improve transmembrane delivery, targeting to specific locations or cell types, increasing interaction with silencing pathway enzymes, increasing release from endosomes).

The iRNAs described herein may contain one or more mismatches with the target sequence. In one embodiment, the iRNA described herein contains three or fewer mismatches. If the antisense strand of the iRNA contains a mismatch with the target sequence, it is preferred that the region of mismatch is not located in the center of the region of complementarity. If the antisense strand of the iRNA contains a mismatch with the target sequence, it is preferred that the mismatch be limited to within the last 5 nucleotides from either the 5' or 3' end of the region of complementarity. For example, for a 23 nucleotide iRNA agent, the strand complementary to the region of the HBV gene generally does not contain any mismatches within the central 13 nucleotides. Methods described herein or known in the art can be used to determine whether an iRNA containing a mismatch with a target sequence is effective in inhibiting expression of an HBV gene. Considering the effectiveness of iRNAs with mismatches in inhibiting HBV gene expression is particularly important when specific regions of complementarity in HBV genes are known to have polymorphic sequence variation within the population. is important.

III. Modified iRNA of the present invention
In one embodiment, the RNA of the iRNA of the invention, e.g., dsRNA, is unmodified, e.g., free of chemical modifications and/or conjugates known in the art and described herein. . In another embodiment, the RNA of the iRNA of the invention, eg, dsRNA, is chemically modified to improve stability or other beneficial properties. In certain embodiments of the invention, substantially all of the nucleotides of the iRNAs of the invention are modified. In other embodiments of the invention, all of the nucleotides of the iRNA of the invention are modified. The iRNA of the present invention in which "substantially all of the nucleotides are modified" means that most of the nucleotides are modified, but not completely modified, with 5 or less, 4 or less, 3 or less, 2 or less , or one or less unmodified nucleotides.

Nucleic acids featured in the present invention are described in "Current protocols in nuclear acid chemistry," Beaucage, S., which is incorporated herein by reference. L. et al. (Edrs.), John Wiley & Sons, Inc. , New York, NY, USA, by well-established methods in the art. Modifications include, for example, terminal modifications, such as 5' end modifications (phosphorylation, conjugates, inverted linkages) or 3' end modifications (conjugates, DNA nucleotides, inverted linkages, etc.); base modifications, e.g. , substitution with stable bases, unstable bases, or bases that base pair with a wide range of partners, removal of bases (non-basic nucleotides), or conjugated bases; sugar modifications (e.g., at the 2' or 4' positions) or sugar substitution; and/or backbone modifications including modification or substitution of phosphodiester bonds. Specific examples of iRNA compounds useful in embodiments described herein include, but are not limited to, RNAs that contain modified backbones or do not contain natural internucleoside linkages. Examples of RNAs having modified skeletons include those that do not have phosphorus atoms in their skeletons. For purposes of this specification, and as sometimes referred to in the art, modified RNAs that do not have phosphorus atoms in the internucleoside backbone can also be considered oligonucleosides. In certain embodiments, the modified iRNA has a phosphorus atom in its internucleoside backbone.

Modified RNA backbones include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkyl phosphotriesters, methyl phosphonates and other alkyl phosphonates, including 3'-alkylene phosphonates and chiral phosphonates, phosphinates, 3' - Phosphoramidates, including aminophosphoramidates and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and with conventional 3'-5' linkages Boranophosphates, their 2'-5' linked analogs, and adjacent pairs of nucleoside units are linked 3'-5' to 5'-3' or 2'-5' to 5'-2' , and those having inverted polarity. Various salts, mixed salts and free acid forms are also included.

Representative US patents teaching the preparation of the above-mentioned phosphorus-containing linkages include, but are not limited to, US Pat. No. 3,687,808; US Pat. No. 4,469,863; 476,301; 5,023,243; 5,177,195; 5,188,897; 5,264,423 5,276,019; 5,278,302; 5,286,717; 5,321,131; 5,399 , 676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; Specification No. 5,476,925; Specification No. 5,519,126; Specification No. 5,536,821; Specification No. 5,541,316; Specification No. 5,550, Specification No. 111; Specification No. 5,563,253; Specification No. 5,571,799; Specification No. 5,587,361; Specification No. 5,625,050; Specification No. 6,028,188; Specification No. 6,124,445; Specification No. 6,160,109; Specification No. 6,169,170; Specification No. 6,172,209 No. 6,239,265; No. 6,277,603; No. 6,326,199; No. 6,346,614; No. 6,346,614; 6,444,423; 6,531,590; 6,534,639; 6,608,035; 6,683,167 Specification; Specification No. 6,858,715; Specification No. 6,867,294; Specification No. 6,878,805; Specification No. 7,015,315; Specification No. 7 , 041,816; 7,273,933; 7,321,029; and U.S. Reissue Patent No. RE39464, the entire contents of each of which is incorporated herein by reference.

Modified RNA backbones that do not contain internal phosphorus atoms include short alkyl or cycloalkyl internucleoside linkages, mixed heteroatoms and alkyl or cycloalkyl internucleoside linkages, or one or more short heteroatom or heterocyclic internucleoside linkages. It has a skeleton formed by bonds. These include those with morpholino bonds (formed in part from sugar moieties of nucleosides); siloxane skeletons; sulfide, sulfoxide and sulfone skeletons; formacetyl and thioformacetyl skeletons; methyleneformacetyl and thioformacetyl skeletons; Included are alkene-containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others with mixed N, O, S, and CH2 component moieties.

Representative U.S. patents teaching the preparation of the oligonucleosides described above include, but are not limited to, U.S. Pat. No. 5,034,506; U.S. Pat. No. 5,166,315; , 444; 5,214,134; 5,216,141; 5, 235,033; 5,64,562; Specification No. 5,264,564; Specification No. 5,405,938; Specification No. 5,434,257; Specification No. 5,466,677; Specification No. 5,470, Specification No. 967; Specification No. 5,489,677; Specification No. 5,541,307; Specification No. 5,561,225; Specification No. 5,596,086; Specification No. 5,602,240; Specification No. 5,608,046; Specification No. 5,610,289; Specification No. 5,618,704; Specification No. 5,623,070 No. 5,663,312; No. 5,633,360; No. 5,677,437; and No. 5,677,439. The entire contents of each of these are incorporated herein by reference.

In other embodiments, suitable RNA mimetics are contemplated for use in iRNAs, where both the sugar and the internucleoside linkage, ie, the backbone of the nucleotide unit, are replaced with novel groups. The base unit is maintained for hybridization with the appropriate nucleic acid target compound. One such oligomeric compound, RNA mimetics, that has been shown to have excellent hybridization properties is called peptide nucleic acid (PNA). In PNA compounds, the sugar backbone of the RNA is replaced with an amide-containing backbone, particularly an aminoethylglycine backbone. The nucleobase is retained and attached directly or indirectly to the aza nitrogen atom of the amide portion of the backbone. Representative U.S. patents teaching the preparation of PNA compounds include, but are not limited to, U.S. Pat. Nos. 5,539,082; 5,714,331; and 5,719,262. The entire contents of each of these are incorporated herein by reference. Additional PNA compounds suitable for use in the iRNA of the invention are described, for example, in Nielsen et al. , Science, 1991, 254, 1497-1500.

Certain embodiments featured in the present invention include RNAs with phosphorothioate backbones and oligonucleosides with heteroatom backbones, particularly the --CH2--NH-- CH2-, --CH2--N(CH3)--O--CH2-- [known as methylene (methylimino) or MMI skeleton], --CH2--O--N(CH3)--CH2 --, --CH2--N(CH3)--N(CH3)--CH2-- and --N(CH3)--CH2--CH2-- [wherein the natural phosphodiester skeleton is -- -O--P--O--CH2--], and the amide skeleton of US Pat. No. 5,602,240 mentioned above. In certain embodiments, the RNAs featured herein have the morpholino backbone structure of US Pat. No. 5,034,506, discussed above.

Modified RNA may also contain one or more substituted sugar moieties. The iRNAs, e.g., dsRNAs featured herein include, in the 2' position, OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S- or N-alkynyl; could be. Exemplary suitable modifications include O[(CH2)nO]mCH3, O(CH2). nOCH3, O(CH2)nNH2, O(CH2)nCH3, O(CH2)nONH2, and O(CH2)nON[(CH2)nCH3)]2, where n and m are from 1 to about 10. be. In other embodiments, the dsRNA is, at the 2' position, C1-C10 lower alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH3, OCN, Cl, Br, CN, CF3, OCF3, SOCH3, SO2CH3, ONO2, NO2, N3, NH2, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, RNA cleavage group, reporter group, intercalator, iRNA drug It includes one of a group that improves the mechanical properties, or a group that improves the pharmacokinetic properties of the iRNA, and other substituents with similar properties. In certain embodiments, the modification is 2'-methoxyethoxy (2'-O--CH2CH2OCH3, also known as 2'-O-(2-methoxyethyl) or 2'-MOE) (Martin et al., Helv.Chim.Acta, 1995, 78:486-504), ie, contains an alkoxy-alkoxy group. Another exemplary modification is 2'-dimethylaminooxyethoxy, also known as 2'-DMAOE, O(CH2)2ON(CH3)2, described herein in the Examples below. group, and 2'-dimethylaminoethoxyethoxy (also known in the art as 2'-O-dimethylaminoethoxyethyl or 2'-DMAEOE), i.e., 2'-O--CH2--O --CH2--N(CH2)2.

Other modifications include 2'-methoxy (2'-OCH3), 2'-aminopropoxy (2'-OCH2CH2CH2NH2) and 2'-fluoro (2'-F). Similar modifications can also be made at other positions in the RNA of an iRNA, particularly at the 3' position of the sugar on the 3' terminal nucleotide or at the 5' position of the 5' terminal nucleotide in a 2'-5' linked dsRNA. iRNAs can also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar. Representative U.S. patents that teach the preparation of such modified sugar structures include, but are not limited to, U.S. Pat. No. 4,981,957; U.S. Pat. No. 5,118,800; Specification No. 5,319,080; Specification No. 5,359,044; Specification No. 5,393,878; Specification No. 5,446,137; Specification No. 5,466,786 No. 5,514,785; No. 5,519,134; No. 5,567,811; No. 5,576,427; Specification No. 5,591,722; Specification No. 5,597,909; Specification No. 5,610,300; Specification No. 5,627,053; Specification No. 5,639,873 Specification; Specification No. 5,646,265; Specification No. 5,658,873; Specification No. 5,670,633; and Specification No. 5,700,920. , some of these have the same owner as this application. The entire contents of each of the above are incorporated herein by reference.

iRNAs may also include nucleobase (often referred to in the art simply as "bases") modifications or substitutions. As used herein, "unmodified" or "natural" nucleobases refer to the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U). including. Modified nucleobases include deoxy-thymine (dT), 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl of adenine and guanine, and others. Alkyl derivatives, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyluracil and cytosine, 6-azouracil, cytosine and thymine, 5 - uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo, especially 5-bromo, 5 - trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-daazaadenine and 3-deazaguanine and 3-deazaadenine and other synthetic and natural nucleobases such as. Additional nucleobases include those disclosed in U.S. Pat. No. 3,687,808, Modified Nucleosides in Biochemistry, Biotechnology and Medicine, Herdewijn, P.; ed. Wiley-VCH, 2008; Concise Encyclopedia Of Polymer Science and Engineering, pp. 858-859, Kroschwitz, J. L, ed. John Wiley & Sons, 1990, Englisch et al. , Angewandte Chemie, International Edition, 1991, 30, 613, and Sanghvi, YS. , Chapter 15, dsRNA Research and Applications, pp. 289-302, Crooke, S. T. and Lebleu, B. , Ed. , CRC Press, 1993. Some of these nucleobases are particularly useful in increasing the binding affinity of the oligomeric compounds featured in this invention. These include 5-substituted pyrimidines, 6-azapyrimidine and N-2, N-6 and 0-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine. The 5-methylcytosine substituent has been shown to increase nucleic acid duplex stability by 0.6-1.2°C (Sanghvi, Y.S., Crooke, S.T. and Lebleu, B. , Eds., dsRNA Research and Applications, CRC Press, Boca Raton, 1993, pp. 276-278), are exemplary base substitutions, especially when combined with the 2'-O-methoxyethyl sugar modification. be.

Representative U.S. patents that teach the preparation of some of the modified nucleobases described above as well as other modified nucleobases include, but are not limited to, U.S. Pat. No. 3,687,808, supra; No. 4,845,205; No. 5,130,30; No. 5,134,066; No. 5,175,273; No. 5,367,066; No. 5,432,272; No. 5,457,187; No. 5,459,255; No. 5,484,908; No. 5,502,177; No. 5,525,711; No. 5,552,540; No. 5,587,469; No. 5,594,121, No. 5,596,091; No. 5,614,617; No. 5,681,941; No. 5,750,692; No. 6,015,886; No. 6,147,200; 6,166,197; 6,222,025; 6,235,887; 6,380,368; 6,528,640; No. 6,639,062; No. 6,617,438; No. 7,045,610; No. 7,427,672; and No. 7,495,088 The entire contents of each of these are incorporated herein by reference.

The RNA of an iRNA can also be modified to include one or more bicyclic sugar moieties. A "bicyclic sugar" is a furanosyl ring modified by a two-atom bridge. A "bicyclic nucleoside" ("BNA") is a nucleoside that has a sugar moiety that includes a bridge that joins two carbon atoms of the sugar ring, thereby forming a bicyclic ring system. In certain embodiments, the bridge connects the 4'-carbon and 2'-carbon of the sugar ring. Thus, in certain embodiments, agents of the invention may include one or more immobilized nucleic acids (LNA). An immobilized nucleic acid is a nucleotide with a modified ribose moiety that includes an additional bridge linking the 2' and 4' carbons. In other words, LNA is a nucleotide containing a bicyclic sugar moiety that includes a 4'-CH2-O-2' bridge. This structure effectively "locks" the ribose in the 3'-endo structural configuration. Addition of immobilized nucleic acids to siRNA has been shown to increase siRNA stability in serum and reduce off-target effects (Elmen, J. et al., (2005) Nucleic Acids Research 33(1) :439-447; Mook, OR. et al., (2007) Mol Canc Ther 6(3):833-843; Grunweller, A. et al., (2003) Nucleic Acids Research 31(12):3185-3193 ). Examples of bicyclic nucleosides for use in the polynucleotides of the invention include, but are not limited to, nucleosides containing a bridge between the 4' and 2' ribosyl ring atoms. In certain embodiments, antisense polynucleotide agents of the invention include one or more bicyclic nucleosides that include a 4'-2' bridge. Examples of such 4'-2' bridged bicyclic nucleosides include, but are not limited to, 4'-(CH2)-O-2' (LNA); 4'-(CH2)-S-2'; 4'-(CH2)2-O-2' (ENA); 4'-CH(CH3)-O-2' (also called "constrained ethyl" or "cEt") and 4'-CH(CH2OCH3)-O -2' (and analogs thereof; see e.g. U.S. Pat. No. 7,399,845); 4'-C(CH3)(CH3)-O-2' (and analogs thereof; e.g. 4'-CH2-N(OCH3)-2' (and analogs thereof; see, e.g., US Pat. No. 8,278,425); 4 '-CH2-O-N(CH3)-2' (see e.g. US Patent Application Publication No. 2004/0171570); 4'-CH2-N(R)-O-2' (where R is H, C1-C12 alkyl), or a protecting group (see, e.g., U.S. Pat. No. 7,427,672); 4'-CH2-C(H)(CH3)-2' (e.g. , Chattopadhyaya et al., J. Org. Chem., 2009, 74, 118-134); and 4'-CH2-C(=CH2)-2' (and analogs thereof; e.g., U.S. Pat. , 278, 426). The entire contents of each of these are incorporated herein by reference.

Additional representative U.S. patents and U.S. patent publications teaching the preparation of immobilized nucleic acid nucleotides include, but are not limited to, U.S. Pat. No. 6,670,461; No. 6,770,748; No. 6,794,499; No. 6,998,484; No. 7,053,207; No. 7,034,133; No. 7,084,125; No. 7,399,845; No. 7,427,672; No. 7,569,686; No. 7,741,457; No. 8,022,193; No. 8,030,467; No. 8,278,425; No. 8,278,426; 8,278,283; U.S. Patent Application Publication No. 2008/0039618; and U.S. Patent Application Publication No. 2009/0012281, the entire contents of each of which are incorporated herein by reference. It is used in

For example, any of the above bicyclic nucleosides can be prepared with one or more stereochemical sugar configurations, including α-L-ribofuranose and β-D-ribofuranose (WO 99/14226). (see brochure).

The RNA of an iRNA can also be modified to include one or more constrained ethyl nucleotides. As used herein, a "constrained ethyl nucleotide" or "cEt" is a fixed nucleic acid that includes a bicyclic sugar moiety that includes a 4'-CH(CH3)-0-2' bridge. In one embodiment, the constrained ethyl nucleotide is in the S configuration, referred to herein as "S-cEt."

iRNAs of the invention may also include one or more "conformationally restricted nucleotides" ("CRNs"). CRN is a nucleotide analog with a linker joining the C2' and C4' carbons of the ribose or the C3 and -C5' carbons of the ribose. CRN locks the ribose ring into a stable configuration and increases hybridization affinity for mRNA. The linker is long enough to place the oxygen in the optimal position for stability and affinity, and to reduce puckering of the ribose ring.

Representative publications teaching the preparation of some of the above CRNs include, but are not limited to, U.S. Patent Application Publication No. 2013/0190383; and PCT Publication No. WO 2013/036868. , the entire contents of each of which are incorporated herein by reference.

One or more of the nucleotides of an iRNA of the invention may also include a hydroxymethyl substituted nucleotide. "Hydroxymethyl-substituted nucleotides" are acyclic 2'-3'-seco-nucleotides, also referred to as "unlocked nucleic acid" ("UNA") modifications.

Representative U.S. patent publications teaching the preparation of UNA include, but are not limited to, U.S. Patent No. 8,314,227; and U.S. Patent Application Publication No. 2013/0096289; and 2011/0313020, the entire contents of each of which are incorporated herein by reference.

Potentially stable modifications to the ends of RNA molecules include N-(acetylaminocaproyl)-4-hydroxyprolinol (Hyp-C6-NHAc), N-(caproyl4-hydroxy Prolinol (Hyp-C6), N-(acetyl-4-hydroxyprolinol (Hyp-NHAc), thymidine-2'-0-deoxythymidine (ether), N-(aminocaproyl)-4-hydroxyprolinol (Hyp-C6-amino), 2-docosanoyl-uridine-3”-phosphate, inverted base dT (idT), etc. Disclosure of this modification is available in PCT Publication No. WO 2011/005861. Seen in the issue pamphlet.

Other modifications of the nucleotides of the iRNAs of the invention include 5' phosphates or 5' phosphate mimetics, such as 5' terminal phosphates or phosphate mimetics to the antisense strand of the RNAi agent. Suitable phosphate mimetics are described, for example, in US Patent Application Publication No. 2012/0157511, the entire contents of which are incorporated herein by reference.

A. Modified iRNA containing the motif of the invention
In certain embodiments of the invention, double-stranded RNAi agents of the invention include, for example, International Publication No. WO 2013/2013, filed on November 16, 2012, each of which is incorporated herein by reference in its entirety. Examples include drugs having chemical modifications disclosed in Pamphlet No. 075035. As shown herein and in PCT Application No. WO 2013/075035, better results show that one or more motifs of three identical modifications on three contiguous nucleotides can be used in the RNAi agent's sense strand and/or antisense strand, particularly at or near the cleavage site. In certain embodiments, the sense and antisense strands of the RNAi agent may be alternatively fully modified. Introduction of these motifs disrupts the modification pattern of the sense and/or antisense strand, if present. The RNAi agent can be optionally conjugated with a GalNAc derivative ligand, eg, on the sense strand. The obtained RNAi agent exhibits superior gene silencing activity.

More particularly, the sense and antisense strands of the double-stranded RNAi agent contain one or more motifs of three identical modifications on three consecutive nucleotides at or near the cleavage site of at least one strand of the RNAi agent. It was surprisingly discovered that the gene silencing activity of RNAi agents was significantly enhanced when completely modified to have the following properties:

Accordingly, the present invention provides double-stranded RNAi agents capable of inhibiting the expression of target genes (ie, HBV genes) in vivo. RNAi agents include sense and antisense strands. Each strand of an RNAi agent can range in length from 12 to 30 nucleotides. For example, each strand may be 14-30 nucleotides long, 17-30 nucleotides long, 25-30 nucleotides long, 27-30 nucleotides long, 17-23 nucleotides long, 17-21 nucleotides long, 17-19 nucleotides long, 19- It can be 25 nucleotides long, 19-23 nucleotides long, 19-21 nucleotides long, 21-25 nucleotides long, or 21-23 nucleotides long.

The sense and antisense strands typically form double-stranded RNA ("dsRNA"), also referred to herein as an "iRNA agent." The double-stranded region of an iRNA agent can be 12-30 nucleotide pairs in length. For example, the double-stranded region is 14-30 nucleotide pairs long, 17-30 nucleotide pairs long, 27-30 nucleotide pairs long, 17-23 nucleotide pairs long, 17-21 nucleotide pairs long, 17-19 nucleotide pairs long, It can be 19-25 nucleotide pairs long, 19-23 nucleotide pairs long, 19-21 nucleotide pairs long, 21-25 nucleotide pairs long, or 21-23 nucleotide pairs long. In another example, the double-stranded region is selected from lengths of 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, and 27 nucleotides.

In one embodiment, the RNAi agent may include one or more overhang regions and/or capping groups at the 3' end, 5' end, or both ends of one or both strands. The overhang is 1 to 6 nucleotides long, for example, 2 to 6 nucleotides long, 1 to 5 nucleotides long, 2 to 5 nucleotides long, 1 to 4 nucleotides long, 2 to 4 nucleotides long, 1 to 3 nucleotides long, 2 to It can be 3 nucleotides long, or 1-2 nucleotides long. Overhangs can be the result of one strand being longer than the other, or the result of two strands of the same length being staggered. The overhang may form a mismatch with the target mRNA, or the overhang may be complementary to the targeted gene sequence, or it may be another sequence. The first and second strands can also be joined by additional bases, eg, to form a hairpin, or by other non-basic linkers.

In one embodiment, the nucleotides in the overhang region of the RNAi agent each independently include, but are not limited to, 2-F, 2'-O-methyl, thymidine (T), 2'-O-methoxyethyl-5 - 2'-sugar modifications such as methyluridine (Teo), 2'-O-methoxyethyladenosine (Aeo), 2'-O-methoxyethyl-5-methylcytidine (m5Ceo), and any combination thereof , modified or unmodified nucleotides. For example, TT can be an overhang sequence for either end on either strand. The overhang may form a mismatch with the target mRNA, or the overhang may be complementary to the targeted gene sequence, or it may be another sequence.

5'- or 3'-overhangs on the sense strand, antisense strand or both strands of the RNAi agent can be phosphorylated. In certain embodiments, the overhang region includes two nucleotides with a phosphorothioate between the two nucleotides, where the two nucleotides can be the same or different. In one embodiment, the overhang is at the 3' end of the sense strand, antisense strand, or both strands. In one embodiment, this 3'-overhang is present in the antisense strand. In one embodiment, this 3'-overhang is present in the sense strand.

An RNAi agent may contain only one overhang that can enhance RNAi interference activity without affecting its overall stability. For example, a single-stranded overhang can be located at the 3' end of the sense strand or at the 3' end of the antisense strand. The RNAi can also have a blunt end located at the 5' end of the antisense strand (or the 3' end of the sense strand) or vice versa. Generally, the antisense strand of RNAi has a nucleotide overhang at the 3' end and is blunt at the 5' end. Without wishing to be bound by theory, asymmetric blunt ends at the 5' end of the antisense strand and the 3' overhang of the antisense strand favor the introduction of the guide strand into the RISC process.

In one embodiment, the RNAi agent is a double ended bluntmer 19 nucleotides long, and the sense strand is a double-ended strand of three consecutive nucleotides at positions 7, 8, and 9 from the 5' end. contains at least one motif of '-F modification. The antisense strand contains at least one motif of three 2'-O-methyl modifications at three consecutive nucleotide positions 11, 12, and 13 from the 5' end.

In another embodiment, the RNAi agent is a blunt-ended duplex 20 nucleotides long, and the sense strand has three 2'-F modifications at three consecutive nucleotide positions 8, 9, and 10 from the 5' end. at least one motif. The antisense strand contains at least one motif of three 2'-O-methyl modifications at three consecutive nucleotide positions 11, 12, and 13 from the 5' end.

In yet another embodiment, the RNAi agent is a blunt-ended duplex 21 nucleotides long, and the sense strand includes three 2'-Fs at three consecutive nucleotide positions 9, 10, and 11 from the 5' end. Contains at least one motif of modification. The antisense strand contains at least one motif of three 2'-O-methyl modifications at three consecutive nucleotide positions 11, 12, and 13 from the 5' end.

In one embodiment, the RNAi agent comprises a 21 nucleotide sense strand and a 23 nucleotide antisense strand, the sense strand having three 2'-F modifications at three consecutive nucleotide positions 9, 10, and 11 from the 5' end. the antisense strand contains at least one motif of three 2'-O-methyl modifications at three consecutive nucleotide positions 11, 12, and 13 from the 5' end; One end is blunt while the other end contains a two nucleotide overhang. Preferably, the two nucleotide overhangs are at the 3' end of the antisense strand.

If the two nucleotide overhangs are at the 3' end of the antisense strand, there may be two phosphorothioate internucleotide linkages between the terminal three nucleotides, and two of the three nucleotides are the overhang nucleotides. , the third nucleotide is the paired nucleotide adjacent to the overhang nucleotide. In one embodiment, the RNAi agent further has two phosphorothioate internucleotide linkages between the terminal three nucleotides at both the 5' end of the sense strand and the 5' end of the antisense strand. In one embodiment, all nucleotides in the sense and antisense strands of the RNAi agent, including nucleotides that are part of the motif, are modified nucleotides. In one embodiment, each residue is independently modified, eg, in alternating motifs, with 2'-O-methyl or 3'-fluoro. Optionally, the RNAi agent further comprises a ligand (preferably GalNAc3).

In one embodiment, the RNAi agent comprises a sense strand and an antisense strand, where the sense strand is 25-30 nucleotide residues long, starting at the 5' terminal nucleotide (position 1) and starting at the 5' terminal nucleotide (position 1). positions ~23 contain at least eight ribonucleotides; the antisense strand is 36 to 66 nucleotide residues long, starting from the 3' terminal nucleotide and at the position paired with positions 1 to 23 of the sense strand. at least eight ribonucleotides to form a duplex; at least the 3' terminal nucleotide of the antisense strand is unpaired with the sense strand and up to 6 consecutive 3' terminal nucleotides are unpaired with the sense strand; the 5' end of the antisense strand contains 10 to 30 contiguous nucleotides that are not paired with the sense strand, thereby forming a 3' single-stranded overhang of 1 to 6 nucleotides; form a single-stranded 5' overhang of 10 to 30 nucleotides; at least the 5' and 3' terminal nucleotides of the sense strand will form a single-stranded 5' overhang of 10 to 30 nucleotides; A base that pairs with a nucleotide of the sense strand, thereby forming a substantially double-stranded region between the sense and antisense strands; is sufficiently complementary to the target RNA along at least 19 ribonucleotides of the antisense strand length to reduce expression of the target gene when introduced into a cell; at least one motif of three 2'-F modifications, where at least one of the motifs is at or near the cleavage site. The antisense strand contains at least one motif of three 2'-O-methyl modifications in three consecutive nucleotides at or near the cleavage site.

In one embodiment, the RNAi agent comprises a sense strand and an antisense strand, and the RNAi agent comprises a first strand having a length of at least 25 and no more than 29 nucleotides, and a first strand having a length of at least 25 and no more than 29 nucleotides, a second strand having a length of 30 nucleotides or less, comprising at least one motif of three 2'-O-methyl modifications in three consecutive nucleotides of; the 5' end forms a blunt end, the second strand is 1 to 4 nucleotides longer than the first strand at its 3' end, the double-stranded region is at least 25 nucleotides long, and the second strand is at least 25 nucleotides long; is sufficiently complementary to the target mRNA along at least 19 nucleotides of the second strand length such that the RNAi agent reduces expression of the target gene when introduced into mammalian cells; Dicer cleavage of the siRNA preferentially results in siRNA containing the 3' end of the second strand, thereby reducing expression of the target gene in the mammal. Optionally, the RNAi agent further comprises a ligand.

In one embodiment, the sense strand of the iRNA agent comprises at least one motif of three identical modifications in three consecutive nucleotides, and one of the motifs is at the cleavage site of the sense strand.

In one embodiment, the antisense strand of the RNAi agent can also include at least one motif of three identical modifications in three consecutive nucleotides, and one of the motifs is at or near the cleavage site of the antisense strand. do.

For RNAi agents with double-stranded regions of 17-23 nucleotides in length, the cleavage site for the antisense strand is typically near positions 10, 11, and 12 from the 5' end. Therefore, three identical modification motifs are present at the first pair in the double-stranded region, starting from the first nucleotide from the 5' end of the antisense strand, or from the 5' end of the antisense strand. Counting from the nucleotide, positions 9, 10, 11 of the antisense strand; 10, 11, 12; 11, 12, 13; 12, 13, 14; or 13, 14 It can exist in the 15th and 15th positions. The cleavage site in the antisense strand may also vary depending on the length of the double-stranded region of RNAi from the 5' end.

The sense strand of the RNAi agent may contain at least one motif of three identical modifications in three consecutive nucleotides at the cleavage site of the strand; the antisense strand may contain at least one motif of three identical modifications in three consecutive nucleotides at or near the cleavage site of the strand. It may have at least one motif of three identical modifications. When the sense and antisense strands form a dsRNA duplex, the sense and antisense strands have at least one motif of three nucleotides in the sense strand and one motif of three nucleotides in the antisense strand. nucleotide overlap, i.e., aligned such that at least one of the three nucleotides of the motif in the sense strand forms a base pair with at least one of the three nucleotides of the motif in the antisense strand. can be done. Alternatively, at least two nucleotides may overlap, or all three nucleotides may overlap.

In one embodiment, the sense strand of the RNAi agent may contain two or more motifs of three identical modifications in three consecutive nucleotides. The first motif may be at or near the strand break site and the other motif may be a wing modification. The term "wing modification" herein refers to a motif that is present on another part of the strand, away from the motif at or near the cleavage site on the same strand. The wing modifications are adjacent to or separated by at least one or more nucleotides from the first motif. When the motifs are directly adjacent to each other, the chemical structures of the motifs are different from each other; when the motifs are separated by one or more nucleotides, the chemical structures can be the same or different. More than one wing modification may be present. For example, if two wing modifications are present, each wing modification may be present at one end or on either side of the lead motif relative to the first motif at or near the cleavage site. .

Similar to the sense strand, the antisense strand of an RNAi agent may contain two or more motifs of three identical modifications in three consecutive nucleotides, with at least one of the motifs at or near the strand cleavage site. exist. The antisense strand may also contain one or more wing modifications with a similar sequence to wing modifications that may be present on the sense strand.

In one embodiment, the wing modification in the sense or antisense strand of the RNAi agent typically does not include the first one or two terminal nucleotides at the 3' end, 5' end, or both ends of the strand. .

In another embodiment, the wing modification in the sense or antisense strand of the RNAi agent is typically within the double-stranded region at the 3' end, 5' end, or both ends of the strand. Contains no two paired nucleotides.

If the sense and antisense strands of the RNAi agent each contain at least one wing modification, the wing modifications may be located at the same end of the double-stranded region, with an overlap of one, two or three nucleotides. may have.

When the sense and antisense strands of the RNAi agent each contain at least two wing modifications, the sense and antisense strands are such that the two modifications from one strand are each located at one end of the double-stranded region. with an overlap of 1, 2 or 3 nucleotides; each of the 2 modifications from one strand is located at the other end of the double-stranded region and has an overlap of 1, 2 or 3 nucleotides; have an overlap of nucleotides; two modifications from one strand can be aligned to have an overlap of 1, 2 or 3 nucleotides in the double-stranded region, located on each side of the lead motif .

In one embodiment, all nucleotides in the sense and antisense strands of the RNAi agent may be modified, including nucleotides that are part of the motif. Each nucleotide may be modified with the same or different modifications, including one or more changes in one or both of the unbound phosphate oxygen and/or the bound phosphate oxygen; a ribose sugar; components, such as modification of the 2' hydroxyl of the ribose sugar; extensive replacement of the phosphate moiety by a "dephospho" linker; modification or substitution of natural bases; and replacement or modification of the ribose-phosphate backbone. may include.

Because nucleic acids are polymers of subunits, many of the modifications, such as modifications of bases or phosphate moieties or non-bonded O's of phosphate moieties, are present at repeated positions within the nucleic acids. In some cases, modifications may be present at all desired positions in the nucleic acid, but in many cases this is not the case. By way of example, modifications may be present only in the 3' or 5' terminal positions, or only in the terminal regions, e.g., positions on the terminal nucleotide or the last 2, 3, 4, 5, or 10 nucleotides of a strand. May exist. Modifications may be present in the double-stranded region, the single-stranded region, or both. Modifications may be present only in double-stranded regions of the RNA, or only in single-stranded regions of the RNA. For example, the phosphorothioate modification at the non-bonded O position may be present only at one or both ends, or at the terminal region, e.g., at the position on the terminal nucleotide or at the last 2, 3, 4, 5, or 10 of the strand. It may be present only in nucleotides or in double-stranded and single-stranded regions, especially at the ends. The 5' end or both ends may be phosphorylated.

For example, increasing stability, including specific bases in the overhang, or adding modified nucleotides or nucleotide surrogates to single-stranded overhangs, e.g., 5' or 3' overhangs, or both. It may be possible to include. For example, it may be desirable to include purine nucleotides in the overhang. In certain embodiments, all or some of the bases in the 3' or 5' overhang may be modified, eg, with the modifications described herein. Modifications include, for example, the use of modifications at the 2' position of the ribose sugar by modifications known in the art, e.g. deoxyribonucleotides, 2'-deoxy-2'-fluoro(2'- F) or the use of 2'-O-methyl modifications, and modifications of phosphate groups, such as phosphorothioate modifications. The overhang need not be homologous to the target sequence.

In one embodiment, each residue in the sense and antisense strands is independently LNA, CRN, cET, UNA, HNA, CeNA, 2'-methoxyethyl, 2'-O-methyl, 2'-O - modified with allyl, 2'-C-allyl, 2'-deoxy, 2'-hydroxyl, or 2'-fluoro. A chain may contain more than one modification. In one embodiment, each residue of the sense and antisense strands is independently modified with 2'-O-methyl or 2'-fluoro.

At least two different modifications are typically present on the sense and antisense strands. Those two modifications may be 2'-O-methyl or 2'-fluoro modifications, or others.

In one embodiment, Na and/or Nb include alternating patterns of modifications. The term "alternating motif" as used herein refers to a motif with one or more modifications, each modification being present on alternate nucleotides of one strand. Alternating nucleotides can refer to every other nucleotide or every third nucleotide, or similar patterns. For example, if A, B, and C each represent one type of modification to a nucleotide, the alternating motifs are "ABABABABABAB...", "AABBAABBAABB...", "AABAABABABAAB...", "AAABAAABAAAB..." ...", "AAABBBAAABBB...", or "ABCABCABCABC...".

The types of modifications included in alternating motifs may be the same or different. For example, if A, B, C, D each represent one type of modification on a nucleotide, then the alternating pattern, i.e. the modification on every other nucleotide, may be the same, but on the sense strand or Each of the antisense strands is selected from several possibilities of modification within alternating motifs such as "ABABAB...", "ACACAC...", "BDBDBD..." or "CDCDCD...". obtain.

In one embodiment, the RNAi agent of the invention comprises a modification pattern of alternating motifs in the sense strand that is shifted relative to a modification pattern of alternating motifs in the antisense strand. The shift may be such that a modified group of a nucleotide in the sense strand corresponds to a different modified group of a nucleotide in the antisense strand, or vice versa. For example, when the sense strand is paired with the antisense strand in a dsRNA duplex, the alternating motifs in the sense strand may start with "ABABAB" from 5' to 3' of the strand, and the antisense Alternating motifs in the strands may begin with "BABABA" from 5' to 3' of the strand within the double-stranded region. As another example, alternating motifs in the sense strand may start with "AABBAABB" from 5' to 3' of the strand, and alternating motifs in the antisense strand may start from 5' to 3' of the strand, and alternating motifs in the antisense strand may ' to 3' may start from 'BBAABBAA', whereby there is a complete or partial shift in the modification pattern between the sense and antisense strands.

In one embodiment, the RNAi agent comprises a pattern of alternating motifs of 2'-O-methyl and 2'-F modifications on the sense strand, which pattern first begins with 2'-O-methyl and 2'-F modifications on the antisense strand. It has a shift to the motif pattern of alternating methyl and 2'-F modifications, i.e., 2'-O-methyl modified nucleotides in the sense strand base pair with 2'-F modified nucleotides in the antisense strand. And vice versa. Position 1 of the sense strand may begin with a 2'-F modification, and position 1 of the antisense strand may begin with a 2'-O-methyl modification.

Introduction of one or more motifs of three identical modifications on three consecutive nucleotides into the sense and/or antisense strand interrupts the initial modification pattern present in the sense and/or antisense strand. do. This disruption of the modification pattern of the sense and/or antisense strands by introducing one or more motifs of three identical modifications on three consecutive nucleotides into the sense and/or antisense strands It unexpectedly increases gene silencing activity against.

In one embodiment, when a motif of three identical modifications on three consecutive nucleotides is introduced into either of the strands, the modification of the nucleotides adjacent to the motif is a different modification than the modification of the motif. For example, a part of a sequence that includes a motif is "...NaYYYNb...", where "Y" represents a modification of the motif of three identical modifications in three consecutive nucleotides, and "Na" and "Nb" represent a modification of the nucleotide adjacent to the motif "YYY" that is different from the modification of Y, and Na and Nb can be the same or different modifications. Alternatively, Na and/or Nb may or may not be present if wing modification is present.

The RNAi agent can further include at least one phosphorothioate or methylphosphonate internucleotide linkage. The phosphorothioate or methylphosphonate internucleotide linkage modification may be present at any nucleotide of the sense or antisense strand or both strands at any position on the strand. For example, an internucleotide bond modification may be present on every nucleotide in the sense and/or antisense strand; each internucleotide bond modification may be present in an alternating pattern in the sense and/or antisense strand. or the sense or antisense strand may contain modifications of both internucleotide linkages in an alternating pattern. The alternating pattern of internucleotide bond modifications in the sense strand may be the same as or different from the antisense strand, and the alternating pattern of internucleotide bond modifications in the sense strand may be the same as the internucleotide bond modifications in the antisense strand. may have a shift to an alternating pattern of . In one embodiment, a double-stranded RNAi agent comprises 6 to 8 phosphorothioate internucleotide linkages. In one embodiment, the antisense strand comprises two phosphorothioate internucleotide linkages at the 5' end and two phosphorothioate internucleotide linkages at the 3' end, and the sense strand comprises at least two phosphorothioate internucleotide linkages at either the 5' end or the 3' end. Contains phosphorothioate internucleotide linkages.

In one embodiment, the RNAi comprises a phosphorothioate or methylphosphonate internucleotide bond modification in the overhang region. For example, an overhang region can include two nucleotides with a phosphorothioate or methylphosphonate internucleotide linkage between the two nucleotides. Internucleotide bond modifications may also be made to join overhanging nucleotides with terminal mating nucleotides within the double-stranded region. For example, at least two, three, four, or all of the overhanging nucleotides may be linked by phosphorothioate or methylphosphonate internucleotide linkages, optionally connecting the overhanging nucleotide to a paired nucleotide adjacent to the overhanging nucleotide. There may be additional phosphorothioate or methylphosphonate internucleotide linkages attached. For example, there may be at least two phosphorothioate internucleotide linkages between the terminal three nucleotides, two of the three nucleotides are overhang nucleotides, and a third nucleotide is adjacent to the overhang nucleotide. Pairing nucleotides. These terminal three nucleotides may be present at the 3' end of the antisense strand, at the 3' end of the sense strand, at the 5' end of the antisense strand, and/or at the 5' end of the antisense strand.

In one embodiment, the two nucleotide overhangs are at the 3' end of the antisense strand, there are two phosphorothioate internucleotide linkages between the terminal three nucleotides, and two of the three nucleotides nucleotide, the third nucleotide being the matching nucleotide adjacent to the overhanging nucleotide. Optionally, the RNAi agent may further have two phosphorothioate internucleotide linkages between the terminal three nucleotides at both the 5' end of the sense strand and the 5' end of the antisense strand.

In one embodiment, the RNAi agent comprises a mismatch with the target, a mismatch within the duplex, or a combination thereof. Mismatches may occur in overhang regions or double-stranded regions. Base pairs can be dissociated or melted (e.g., with respect to the free energy of binding or dissociation of a particular pair, and the simplest approach is to examine each pair individually, but similar or similar analysis may also be used). Regarding promotion of dissociation: A:U is preferred over G:C; G:U is preferred over G:C; I:C is preferred over G:C (I=inosine). Mismatches, e.g. non-canonical or non-canonical pairings (as described elsewhere herein) are preferred over canonical (A:T, A:U, G:C) pairings; Pairings involving universal bases are preferred over canonical pairings.

In one embodiment, the RNAi agent is the first one, two or more within the double-stranded region from the 5' end of the antisense strand independently selected from the group A:U, G:U, I:C. at least one of three, four, or five base pairs, and a mismatched pair to promote dissociation of the antisense strand at the 5' end of the duplex, e.g., non-canonical or canonical. This includes pairings other than or including universal bases.

In one embodiment, the nucleotide at position 1 within the double-stranded region from the 5' end of the antisense strand is selected from the group consisting of A, dA, dU, U, and dT. Alternatively, at least one of the first 1, 2 or 3 base pairs within the double-stranded region from the 5' end of the antisense strand is an AU base pair. For example, the first base pair within the double-stranded region from the 5' end of the antisense strand is an AU base pair.

In another embodiment, the nucleotide at the 3' end of the sense strand is deoxythymine (dT). In another embodiment, the 3' terminal nucleotide of the antisense strand is deoxythymine (dT). In one embodiment, there is a short sequence of deoxythymine nucleotides, such as two dT nucleotides, at the 3' end of the sense and/or antisense strand.

In one embodiment, the sense strand sequence has the formula (I):
5'np-Na-(XXX)i-Nb-YYY-Nb-(ZZZ)j-Na-nq3'(I)
(In the formula:
i and j are each independently 0 or 1;
p and q are each independently 0 to 6;
each Na independently represents an oligonucleotide sequence comprising from 0 to 25 modified nucleotides, each sequence comprising at least two differently modified nucleotides;
each Nb independently represents an oligonucleotide sequence comprising 0 to 10 modified nucleotides;
each np and nq independently represents an overhanging nucleotide;
where Nb and Y do not have the same modification;
XXX, YYY and ZZZ each independently represent one motif of three identical modifications in three consecutive nucleotides)
can be represented by Preferably, YYY are all 2'-F modified nucleotides.

In one embodiment, Na and/or Nb include alternating patterns of modifications.

In one embodiment, the YYY motif is at or near the cleavage site of the sense strand. For example, if the RNAi agent has a double-stranded region between 17 and 23 nucleotides in length, the YYY motif can be added starting from the first nucleotide from the 5' end; may be at or near the cleavage site of the sense strand, counting from the first paired nucleotide in the region (for example: positions 6, 7, 8, 7, 8, 9, 8, 9, 10, 9, 10, 11, 10, 11, 12 or 11, 12, 13).

In one embodiment, i is 1 and j is 0, or i is 0 and j is 1, or both i and j are 1. Thus, the sense strand may be represented by the following formula:
5'np-Na-YYY-Nb-ZZZ-Na-nq3'(Ib);
5'np-Na-XXX-Nb-YYY-Na-nq3'(Ic); or 5'np-Na-XXX-Nb-YYY-Nb-ZZZ-Na-nq3' (Id).

When the sense strand is represented by formula (Ib), Nb represents an oligonucleotide sequence comprising 0-10, 0-7, 0-5, 0-4, 0-2 or 0 modified nucleotides. Each Na can independently represent an oligonucleotide sequence containing 2-20, 2-15, or 2-10 modified nucleotides.

When the sense strand is represented by formula (Ic), Nb is 0-10, 0-7, 0-10, 0-7, 0-5, 0-4, 0-2 or 0 modified nucleotides. represents an oligonucleotide sequence containing Each Na can independently represent an oligonucleotide sequence containing 2-20, 2-15, or 2-10 modified nucleotides.

When the sense strand is represented by formula (Id), each Nb independently comprises an oligonucleotide comprising 0-10, 0-7, 0-5, 0-4, 0-2 or 0 modified nucleotides. Represents an array. Preferably, Nb is 0, 1, 2, 3, 4, 5 or 6. Each Na can independently represent an oligonucleotide sequence containing 2-20, 2-15, or 2-10 modified nucleotides.

Each of X, Y and Z can be the same or different from each other.

In other embodiments, i is 0 and j is 0, and the sense strand can be represented by the following formula:
5'np-Na-YYY-Na-nq3' (Ia).

When the sense strand is represented by formula (Ia), each Na can independently represent an oligonucleotide sequence comprising 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

In one embodiment, the RNAi antisense strand sequence has the formula (II):
5'nq'-Na'-(Z'Z'Z')k-Nb'-Y'Y'Y'-Nb'-(X'X'X')l-N'a-np'3'( II)
(In the formula:
k and l are each independently 0 or 1;
p' and q' are each independently 0 to 6;
each Na' independently represents an oligonucleotide sequence comprising from 0 to 25 modified nucleotides, each sequence comprising at least two differently modified nucleotides;
each Nb' independently represents an oligonucleotide sequence comprising 0 to 10 modified nucleotides;
each np' and nq' independently represents an overhanging nucleotide;
where Nb' and Y' do not have the same modification;
X'X'X', Y'Y'Y' and Z'Z'Z' each independently represent one motif of three identical modifications in three consecutive nucleotides)
can be represented by

In one embodiment, Na' and/or Nb' include alternating patterns of modifications.

The Y'Y'Y' motif is present at or near the cleavage site of the antisense strand. For example, if the RNAi agent has a double-stranded region between 17 and 23 nucleotides in length, the Y'Y'Y' motif may be added starting from the first nucleotide from the 5' end; Starting from the first paired nucleotide in the double-stranded region, positions 9, 10, and 11 of the antisense strand; positions 10, 11, and 12; positions 11, 12, and 13; 12th, 13th, 14th; or may be present at 13th, 14th, 15th. Preferably, the Y'Y'Y' motif is present at positions 11, 12, and 13.

In one embodiment, the Y'Y'Y' motifs are all 2'-OMe modified nucleotides.

In one embodiment, k is 1 and l is 0, or k is 0 and l is 1, or both k and l are 1.

Thus, the antisense strand may be represented by the following formula:
5'nq'-Na'-Z'Z'Z'-Nb'-Y'Y'Y'-Na'-np'3'(IIb);
5'nq'-Na'-Y'Y'Y'-Nb'-X'X'X'-np'3'(IIc); or 5'nq'-Na'-Z'Z'Z'-Nb '-Y'Y'Y'-Nb'-X'X'X'-Na'-np'3' (IId).

When the antisense strand is represented by formula (IIb), Nb' is 0-10, 0-7, 0-10, 0-7, 0-5, 0-4, 0-2 or 0 modification Represents an oligonucleotide sequence containing nucleotides. Each Na' independently represents an oligonucleotide sequence containing 2-20, 2-15, or 2-10 modified nucleotides.

When the antisense strand is represented by formula (IIc), Nb' is 0-10, 0-7, 0-10, 0-7, 0-5, 0-4, 0-2 or 0 modification Represents an oligonucleotide sequence containing nucleotides. Each Na' independently represents an oligonucleotide sequence containing 2-20, 2-15, or 2-10 modified nucleotides.

When the antisense strand is represented by formula (IId), each Nb' is independently 0-10, 0-7, 0-10, 0-7, 0-5, 0-4, 0- Represents an oligonucleotide sequence containing 2 or 0 modified nucleotides. Each Na' independently represents an oligonucleotide sequence containing 2-20, 2-15, or 2-10 modified nucleotides. Preferably, Nb is 0, 1, 2, 3, 4, 5 or 6.

In other embodiments, k is 0 and l is 0, and the antisense strand can be represented by the formula:
5'np'-Na'-Y'Y'Y'-Na'-nq'3' (Ia).

When the antisense strand is represented as formula (IIa), each Na' independently represents an oligonucleotide sequence comprising 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

Each of X', Y' and Z' can be the same or different from each other.

Each nucleotide in the sense and antisense strands is independently LNA, CRN, UNA, cEt, HNA, CeNA, 2'-methoxyethyl, 2'-O-methyl, 2'-O-allyl, 2'- Can be modified with C-allyl, 2'-hydroxyl, or 2'-fluoro. For example, each nucleotide of the sense and antisense strands is independently modified with 2'-O-methyl or 2'-fluoro. Each X, Y, Z, X', Y' and Z' may represent, inter alia, a 2'-O-methyl modification or a 2'-fluoro modification.

In one embodiment, if the double-stranded region is 21 nucleotides, the sense strand of the RNAi agent starts from the first nucleotide from the 5' end; It may include a YYY motif present at positions 9, 10, and 11 of the strand, counting from the first paired nucleotide within the region; Y represents a 2'-F modification. The sense strand may further include a XXX motif or a ZZZ motif as a wing modification at the opposite end of the double-stranded region; XXX and ZZZ each independently represent a 2'-OMe modification or a 2'-F Represents a modification.

In one embodiment, the antisense strand is counted starting from the first nucleotide from the 5' end; or optionally starting from the first paired nucleotide within the double-stranded region from the 5' end; It may contain a Y'Y'Y' motif present at positions 11, 12, and 13 of the chain; Y' represents a 2'-O-methyl modification. The antisense strand may further include an X'X'X' motif or a Z'Z'Z' motif as a wing modification at the opposite end of the double-stranded region; X'X'X' and Z' Each Z'Z' independently represents a 2'-OMe modification or a 2'-F modification.

The sense strand represented by any one of formulas (Ia), (Ib), (Ic), and (Id) above is represented by formulas (IIa), (IIb), (IIc), and (IId), respectively. forms a double strand with the antisense strand represented by any one of the following.

Accordingly, RNAi agents for use in the methods of the invention may include a sense strand and an antisense strand, each strand having 14-30 nucleotides, and the RNAi duplex having the formula (III ):
Sense: 5'np-Na-(XXX)i-Nb-YYY-Nb-(ZZZ)j-Na-nq3'
Antisense: 3'np'-Na'-(X'X'X')k-Nb'-Y'Y'Y'-Nb'-(Z'Z'Z')l-Na'-nq'5 '
(III)
(In the formula:
i, j, k, and l are each independently 0 or 1;
p, p', q, and q' are each independently 0 to 6;
each Na and Na' independently represents an oligonucleotide sequence comprising 0 to 25 modified nucleotides, each sequence comprising at least two differently modified nucleotides;
each Nb and Nb' independently represents an oligonucleotide sequence comprising 0 to 10 modified nucleotides;
here,
each np', np, nq', and nq, each of which may or may not be present, independently represents an overhanging nucleotide;
XXX, YYY, ZZZ, X'X'X', Y'Y'Y', and Z'Z'Z' each independently represent one motif of three identical modifications on three consecutive nucleotides )
Represented by

In one embodiment, i is 0 and j is 0; or i is 1 and j is 0; or i is 0 and j is 1; or both i and j are 0; or both i and j are 1. In another embodiment, k is 0 and l is 0; or k is 1 and l is 0; k is 0 and l is 1; or both k and l are 0; or both k and l are 1.

Exemplary combinations of sense and antisense strands that form RNAi duplexes include the following formulas:
5'np-Na-YYY-Na-nq3'
3'np'-Na'-Y'Y'Y'-Na'nq'5'
(IIIa)
5'np-Na-YYY-Nb-ZZZ-Na-nq3'
3'np'-Na'-Y'Y'Y'-Nb'-Z'Z'Z'-Na'nq'5'
(IIIb)
5'np-Na-XXX-Nb-YYY-Na-nq3'
3'np'-Na'-X'X'X'-Nb'-Y'Y'Y'-Na'-nq'5'
(IIIc)
5'np-Na-XXX-Nb-YYY-Nb-ZZZ-Na-nq3'
3'np'-Na'-X'X'X'-Nb'-Y'Y'Y'-Nb'-Z'Z'Z'-Na-nq'5'
(IIId)

When the RNAi agent is represented by formula (IIIb), each Nb independently represents an oligonucleotide sequence comprising 1-10, 1-7, 1-5 or 1-4 modified nucleotides. Each Na independently represents an oligonucleotide sequence containing 2-20, 2-15, or 2-10 modified nucleotides.

When the RNAi agent is represented by formula (IIIc), each Nb, Nb' is independently 0-10, 0-7, 0-10, 0-7, 0-5, 0-4, 0- Represents an oligonucleotide sequence containing 2 or 0 modified nucleotides. Each Na independently represents an oligonucleotide sequence containing 2-20, 2-15, or 2-10 modified nucleotides.

When the RNAi agent is represented by formula (IIId), each Nb, Nb' is independently 0-10, 0-7, 0-10, 0-7, 0-5, 0-4, 0- Represents an oligonucleotide sequence containing 2 or 0 modified nucleotides. Each Na, Na' independently represents an oligonucleotide sequence containing 2-20, 2-15, or 2-10 modified nucleotides. Each of Na, Na', Nb and Nb' independently includes alternating patterns of modifications.

When the iRNA agent is represented by formula (IIId), each Nb, Nb' is independently 0-10, 0-7, 0-10, 0-7, 0-5, 0-4, 0 Represents an oligonucleotide sequence containing ~2 or 0 modified nucleotides. Each Na, Na' independently represents an oligonucleotide sequence containing 2-20, 2-15, or 2-10 modified nucleotides. Each of Na, Na', Nb and Nb' independently includes alternating patterns of modifications. Each of X, Y and Z in formulas (III), (IIIa), (IIIb), (IIIc) and (IIId) may be the same or different from each other.

When the RNAi agent is represented by formulas (III), (IIIa), (IIIb), (IIIc), and (IIId), at least one of the Y nucleotides may base pair with one of the Y' nucleotides. . Alternatively, at least two of the Y nucleotides base pair with the corresponding Y' nucleotide; or all three of the Y nucleotides base pair with the corresponding Y' nucleotide.

When the RNAi agent is represented by formula (IIIb) or (IIId), at least one of the Z nucleotides may base pair with one of the Z' nucleotides. Alternatively, at least two of the Z nucleotides base pair with the corresponding Z' nucleotide; or all three of the Z nucleotides base pair with the corresponding Z' nucleotide.

When the RNAi agent is represented by formula (IIIc) or (IIId), at least one of the X nucleotides may base pair with one of the X' nucleotides. Alternatively, at least two of the X nucleotides base pair with the corresponding X' nucleotide; or all three of the X nucleotides base pair with the corresponding X' nucleotide.

In one embodiment, the modification on the Y nucleotide is different from the modification on the Y' nucleotide, the modification on the Z nucleotide is different from the modification on the Z' nucleotide, and/or the modification on the X nucleotide is different from the modification on the X' nucleotide. Different from the above modification.

In one embodiment, when the RNAi agent is represented by formula (IIId), the Na modification is a 2'-O-methyl or 2'-fluoro modification. In another embodiment, when the RNAi agent is represented by formula (IIId), the Na modification is a 2'-O-methyl or 2'-fluoro modification, np'>0, and at least one np ' is attached to adjacent nucleotides via phosphorothioate bonds. In yet another embodiment, when the RNAi agent is represented by formula (IIId), the Na modification is a 2'-O-methyl or 2'-fluoro modification, np'>0, and at least one np' is attached to adjacent nucleotides via a phosphorothioate bond, and the sense strand is conjugated to one or more GalNAc derivatives attached via a divalent or trivalent branched linker ( (described below). In another embodiment, when the RNAi agent is represented by formula (IIId), the Na modification is a 2'-O-methyl or 2'-fluoro modification, np'>0, and at least one np ' is linked to adjacent nucleotides via phosphorothioate linkages, the sense strand comprises at least one phosphorothioate linkage, and the sense strand contains one linked via a divalent or trivalent branched linker. or conjugated to multiple GalNAc derivatives.

In one embodiment, when the RNAi agent is represented by formula (IIIa), the Na modification is a 2'-O-methyl or 2'-fluoro modification, np'>0, and at least one np' is linked to adjacent nucleotides via phosphorothioate linkages, the sense strand includes at least one phosphorothioate linkage, and the sense strand includes one or more nucleotides linked via a divalent or trivalent branched linker. Conjugated to multiple GalNAc derivatives.

In one embodiment, the RNAi agent is a multimer comprising at least two duplexes represented by formulas (III), (IIIa), (IIIb), (IIIc), and (IIId); The chains are joined by linkers. Linkers can be cleavable or non-cleavable. Optionally, the multimer further comprises a ligand. Each of the duplexes can target the same gene or two different genes; or each of the duplexes can target the same gene at two different target sites.

In one embodiment, the RNAi agent comprises three, four, five, six or more dinucleotides represented by formulas (III), (IIIa), (IIIb), (IIIc), and (IIId). It is a multimer containing two strands, and the two strands are joined by a linker. Linkers can be cleavable or non-cleavable. Optionally, the multimer further comprises a ligand. Each of the duplexes can target the same gene or two different genes; or each of the duplexes can target the same gene at two different target sites.

In one embodiment, the two RNAi agents represented by formulas (III), (IIIa), (IIIb), (IIIc), and (IIId) are attached to each other at one or both of the 5' and 3' ends. attached and optionally conjugated to a ligand. Each of the RNAi agents can target the same gene or two different genes; or each of the RNAi agents can target the same gene at two different target sites.

Various publications describe multimeric RNAi agents that can be used in the methods of the invention. Such publications include WO 2007/091269 pamphlet, US Patent No. 7858769, WO 2010/141511 pamphlet, WO 2007/117686 pamphlet, and WO 2009/014887. pamphlet and WO 2011/031520 pamphlet, the entire contents of each of which are incorporated herein by reference.

As explained in more detail below, an RNAi agent that includes conjugation of one or more carbohydrate moieties to the RNAi agent can optimize one or more properties of the RNAi agent. Often, carbohydrate moieties are attached to modified subunits of RNAi agents. For example, the ribose sugar of one or more ribonucleotide subunits of a dsRNA agent can be replaced with another moiety, such as a non-carbohydrate (preferably cyclic) carrier to which a carbohydrate ligand is attached. Ribonucleotide subunits in which the ribose sugar of the subunit has been replaced in this manner are referred to herein as ribose replacement modified subunits (RRMS). Cyclic carriers may be carbocyclic, i.e., all ring atoms are carbon atoms, or heterocyclic, i.e., one or more ring atoms are heteroatoms, e.g. nitrogen, oxygen. , can be sulfur. A cyclic carrier may be a single ring system or may contain two or more rings, such as fused rings. A cyclic carrier may be a fully saturated ring system or may contain one or more double bonds.

A ligand can be attached to a polynucleotide via a carrier. The carrier comprises (i) at least one "backbone attachment point", preferably two "backbone attachment points" and (ii) at least one "tethering attachment point". "Backbone point of attachment" as used herein refers to a ribonucleic acid containing a functional group, e.g., a hydroxyl group, or a backbone, e.g., a phosphate, or a modified phosphate, e.g., sulfur. Refers to bonds available and suitable for the incorporation of carriers into the framework of A "tethered point of attachment" (TAP) refers, in certain embodiments, to a constituent ring atom of a cyclic carrier that connects selected moieties, such as a carbon atom or a heteroatom (different from the atom that provides the backbone point of attachment). This moiety can be, for example, a carbohydrate, such as monosaccharides, di-saccharides, trisaccharides, tetrasaccharides, oligosaccharides and polysaccharides. Optionally, the selected portions are connected to the annular carrier by intervening tethers. Thus, cyclic carriers often contain functional groups, such as amino groups, or generally provide suitable bonds for incorporation or tethering of another chemical moiety, such as a ligand, to the constituent ring.

The iRNA agent may be conjugated to the ligand via a carrier, which may be a cyclic group or a cyclic group; preferably the cyclic group is pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, Preferably the cyclic group is selected from piperidinyl, piperazinyl, [1,3]dioxolane, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridazinonyl, tetrahydrofuryl and decalin; , serinol skeleton or diethanolamine skeleton.

In certain embodiments, the iRNA agent for use in the methods of the invention is listed in any one of Tables 3, 4, 6, 7, 12, 13, 22, 23, 25, and 26. A drug selected from the group of drugs. These agents may further include a ligand.

IV. iRNA conjugated to a ligand
Another modification of the RNA of the iRNA of the invention involves chemically attaching to the RNA one or more ligands, moieties or conjugates that enhance the activity, cellular distribution or cellular uptake of the iRNA. Such moieties include, but are not limited to, cholesterol moieties (Letsinger et al., Proc. Natl. Acid. Sci. USA, 1989, 86:6553-6556), cholic acid (Manoharan et al., Biorg. Med. Chem. Let., 1994, 4: 1053-1060), thioethers such as beryl-S-tritylthiol (Manoharan et al., Ann. NY Acad. Sci., 1992, 660: 306-309; Manoharan et al., Biorg. Med. Chem. Let., 1993, 3: 2765-2770), thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20: 533-538), aliphatic chain, For example, dodecanediol or undecyl residues (Saison-Behmoaras et al., EMBO J, 1991, 10:1111-1118; Kabanov et al., FEBS Lett., 1990, 259:327-330; Svinarchuk et al., B iochimie , 1993, 75:49-54), phospholipids such as di-hexadecyl-rac-glycerol or triethyl-ammonium 1,2-di-O-hexadecyl-rac-glycero-3-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36: 3651-3654; Shea et al., Nucl. Acids Res., 1990, 18: 3777-3783), polyamine or polyethylene glycol chains (Manoharan et al., Nucleosides & Nucleotides, 19 95,14:969- 973), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36: 3651-3654), palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264: 229-237) ), or octadecyl Lipid moieties such as amine or hexylamino-carbonyloxycholesterol moieties (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277:923-937) are included.

In one embodiment, the ligand alters the distribution, targeting, or lifetime of the iRNA agent into which it is incorporated. In a preferred embodiment, the ligand targets a selected target (e.g., molecule, cell or cell type), compartment (e.g., a cell or organ compartment), a target of the body, e.g. compared to a species without such ligand. Provides improved affinity for tissues, organs or areas. Preferred ligands do not participate in duplex pairing in double-stranded nucleic acids.

The ligand can be a protein (e.g., human serum albumin (HSA), low-density lipoprotein (LDL), or globulin); a carbohydrate (e.g., dextran, pullulan, chitin, chitosan, inulin, cyclodextrin, N-acetylgalactosamine, or hyaluronic acid); ); or may contain natural substances such as lipids. Ligands can also be recombinant or synthetic molecules, such as synthetic polymers, eg, synthetic polyamino acids. Examples of polyamino acids include polylysine (PLL), poly-L-aspartic acid, poly-L-glutamic acid, styrene-maleic anhydride copolymer, poly(L-lactide-co-glycolide) copolymer, divinyl ether- maleic anhydride copolymer, N-(2-hydroxypropyl)methacrylamide copolymer (HMPA), polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethane, poly(2-ethyl acrylic acid), N-isopropylacrylamide polymer, or It is polyphosphazine. Examples of polyamines include polyethyleneimine, polylysine (PLL), spermine, spermidine, polyamines, peptidomimetic polyamines, peptidomimetic polyamines, dendrimer polyamines, arginine, amidine, protamine, cationic lipids, cationic porphyrins, quaternary polyamines. salts, or α-helical peptides.

Ligands may also include targeting groups, such as cell or tissue targeting agents, such as lectins, glycoproteins, lipids or proteins, such as antibodies that bind to particular cell types, such as renal cells. Target groups include thyrotropin, melanotropin, lectin, glycoprotein, surfactant protein A, mucin carbohydrate, polyvalent lactose, polyvalent galactose, N-acetyl-galactosamine, N-acetyl-glucosamine, polyvalent mannose, polyvalent Fucose, glycosylated polyamino acids, polygalactose, transferrin, bisphosphonates, polyglutamates, polyaspartates, lipids, cholesterol, steroids, bile acids, folates, vitamin B12, vitamin A, biotin, or RGD peptides, or RGD peptidomimetics Or it can be an aptamer.

Other examples of ligands include dyes, intercalators (e.g. acridine), crosslinkers (e.g. psoralen, mitomycin C), porphyrins (TPPC4, texaphyrin, sapphirin), polycyclic aromatic hydrocarbons (e.g. phenazine, dihydrophenazine). ), artificial endonucleases or chelating agents (e.g. EDTA), lipophilic molecules such as cholesterol, cholic acid, adamantane acetic acid, 1-pyrenebutyric acid, dihydrotestosterone, 1,3-bis-O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid, O3-(oleoyl) lithocholic acid, O3-(oleoyl) cholenic acid, dimethoxytrityl , or phenoxazine) and peptide conjugates (e.g., antennapedia peptide, Tat peptide), alkylating agents, phosphates, amino, mercapto, PEG (e.g., PEG-40K), MPEG, [MPEG]2, polyamino , alkyls, substituted alkyls, radiolabeled markers, enzymes, haptens (e.g. biotin), transport/absorption enhancers (e.g. aspirin, vitamin E, folic acid), synthetic ribonucleases (e.g. imidazole, bisimidazole, histamine, imidazole cluster). ), acridine-imidazole complex, Eu3+tetraaza macrocycle complex), dinitrophenyl, HRP, or AP.

The ligand can be a protein, eg, a glycoprotein, or a peptide, eg, a co-ligand, or an antibody, eg, a molecule that has a specific affinity for antibodies that bind to a particular cell type, such as hepatocytes. Ligands may also include hormones and hormone receptors. Ligands can also include lipids, lectins, carbohydrates, vitamins, cofactors, polylactose, polygalactose, N-acetyl-galactosamine, N-acetyl-glucosamine, or non-peptide species such as polymannose, polyfucose or aptamers. may include. The ligand can be, for example, a lipopolysaccharide, an activator of 38MAP kinase, or an activator of NF-κB.

A ligand is a substance that can enhance the uptake of an iRNA agent into a cell, e.g., by disrupting the cell's microtubules, microfilaments, and/or intermediate filaments, e.g., by disrupting the cytoskeleton, e.g. , can be a drug. The drug can be, for example, taxon, vincristine, vinblastine, cytochalasin, nocodazole, jasplakinolide, latrunculin A, phalloidin, swinholide A, indanosin, or myoserbin.

In certain embodiments, a ligand attached to an iRNA described herein serves as a pharmacokinetic modulator (PK modulator). PK modulators include lipophiles, bile acids, steroids, phospholipid analogs, peptides, protein binders, PEG, vitamins, and the like. Exemplary PK modulators include, but are not limited to, cholesterol, fatty acids, cholic acid, lithocholic acid, dialkylglycerides, diacylglycerides, phospholipids, sphingolipids, naproxen, ibuprofen, vitamin E, biotin, and the like. Oligonucleotides containing several phosphorothioate linkages are also known to bind to serum proteins, and therefore short oligonucleotides, e.g., about 5 bases, 10 bases, 15 bases, containing multiple phosphorothioate linkages in the backbone. Base or 20 base oligonucleotides are also suitable for the present invention as ligands (eg PK modulating ligands). Additionally, aptamers that bind serum components (eg, serum proteins) are also suitable for use as PK modulating ligands in the embodiments described herein.

Ligand-conjugated oligonucleotides of the invention can be synthesized by the use of oligonucleotides with reactive pendant functional groups, such as those derived from attachment of a binding molecule to the oligonucleotide (described below). This reactive oligonucleotide may be reacted directly with a commercially available ligand, a synthesized ligand with any of a variety of protecting groups, or a ligand to which a binding moiety is attached.

Oligonucleotides used in the conjugates of the invention can be conveniently and routinely made by the well-known technique of solid phase synthesis. Equipment for such synthesis is sold by several vendors, including, for example, Applied Biosystems (Foster City, Calif.). Any other means for such synthesis known in the art may be used in addition or in place. It is also known to use similar techniques to prepare other oligonucleotides such as phosphorothioate and alkylated derivatives.

In the ligand-conjugated oligonucleotides and ligand molecules having sequence-specific binding nucleosides of the present invention, the oligonucleotides and oligonucleosides are standard nucleotide or nucleoside precursors, or nucleotide or nucleoside conjugate precursors that already have a binding moiety; Ligand-nucleotide or nucleoside conjugate precursors that already have a ligand molecule, or non-nucleoside ligand-containing building blocks, can be used to assemble in a suitable DNA synthesizer.

When using a nucleotide conjugate precursor that already has a binding moiety, the synthesis of the sequence-specific binding nucleoside is typically completed before the ligand molecule is reacted with the binding moiety to form the ligand-conjugated oligonucleotide. be done. In certain embodiments, oligonucleotides or linked nucleosides of the invention are derived from ligand-nucleoside conjugates in addition to commercially available standard and non-standard phosphoramidites commonly used for oligonucleotide synthesis. Synthesized by an automated synthesizer using derivatized phosphoramidites.

A. Lipid Conjugates In one embodiment, the ligand or conjugate is a lipid or lipid-based molecule. Such lipids or lipid-based molecules preferably bind to serum proteins, such as human serum albumin (HSA). The HSA-binding ligand allows for distribution of the conjugate to target tissues of the body, such as non-kidney target tissues. For example, the target tissue can be the liver, including liver parenchymal cells. Other molecules capable of binding HSA can also be used as ligands. For example, naproxen or aspirin can be used. The lipid or lipid-based ligand (a) increases the resistance of the conjugate to degradation, (b) increases targeting or transport to target cells or cell membranes, and/or (c) inhibits serum proteins, e.g., HSA. can be used to coordinate binding to.

Lipid-based ligands can be used to inhibit, eg, control, binding of the conjugate to target tissues. For example, lipids or lipid-based ligands that bind HSA more strongly are less likely to be targeted to the kidneys and therefore less likely to be cleared from the body. Conjugates can be targeted to the kidney using lipids or lipid-based ligands that bind HSA more weakly.

In a preferred embodiment, the lipid-based ligand binds HSA. Preferably, the lipid-based ligand binds HSA with sufficient affinity such that the conjugate is preferably distributed to non-kidney tissues. However, the affinity is preferably not so strong that HSA-ligand binding cannot be reversed.

In another preferred embodiment, the lipid-based ligand binds weakly or not at all to HSA, such that the conjugate is preferably distributed to the kidneys. Other moieties that target renal cells can also be used in place of or in addition to lipid-based ligands.

In another embodiment, the ligand is a moiety, eg, a vitamin, that is taken up by the target cell, eg, a proliferating cell. These are particularly useful, for example, in treating disorders characterized by unwanted cell proliferation, malignant or non-malignant, such as cancer cells. Exemplary vitamins include vitamins A, E, and K. Other exemplary vitamins include B vitamins, such as folic acid, B12, riboflavin, biotin, pyridoxal or other vitamins or nutrients that are taken up by target cells such as liver cells. Also included are HAS and low density lipoprotein (LDL).

B. Cell-permeation agents In another embodiment, the ligand is a cell-permeation agent, preferably a helical cell-permeation agent. Preferably the agent is amphiphilic. An exemplary agent is a peptide such as tat or antenopedia. If the agent is a peptide, it may be modified including the use of peptidyl mimetics, inverse isomers, non-peptide or pseudo-peptide bonds, and D-amino acids. The helical agent is preferably an α-helical agent, which preferably has a lipophilic and a lipophobic phase.

A ligand can be a peptide or a peptidomimetic. Peptidomimetics (also referred to herein as oligopeptide mimetics) are molecules that can fold into well-defined three-dimensional structures similar to natural peptides. Binding of peptides and peptidomimetics to iRNA agents can affect the pharmacokinetic distribution of the iRNA, eg, by enhancing cellular recognition and absorption. The peptide or peptidomimetic moiety is about 5-50 amino acids long, such as about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids long. obtain.

The peptide or peptidomimetic can be, for example, a cell-penetrating peptide, a cationic peptide, an amphipathic peptide, or a hydrophobic peptide (eg, consisting primarily of Tyr, Trp or Phe). The peptide moiety can be a dendrimeric peptide, a structurally constrained peptide or a cross-linked peptide. In another alternative, the peptide moiety may include a hydrophobic membrane transport sequence (MTS). An exemplary hydrophobic MTS-containing peptide is RFGF, which has the amino acid sequence AAVALLPAVLLALLAP (SEQ ID NO: 43). RFGF analogs containing hydrophobic MTS (e.g., amino acid sequence AALLPVLLAAP (SEQ ID NO: 44)) can also be targeting moieties. Peptide moieties transport large polar molecules including peptides, oligonucleotides, and proteins across cell membranes. For example, sequences derived from the HIV Tat protein (GRKKRRQRRRPPQ (SEQ ID NO: 45) and Drosophila Antennapedia protein (RQIKIWFQNRRMKWKK (SEQ ID NO: 46)) can function as delivery peptides. Peptides or peptidomimetics can be identified from phage display libraries or from one-bead-one-compound (OBOC) combinatorial libraries. (Lam et al., Nature, 354:82-84, 1991). Peptides or peptides linked to a dsRNA agent via monomer units incorporated for the purpose of cellular targeting. An example of a mimetic is a peptide such as arginine-glycine-aspartic acid (RGD)-peptide, or an RGD mimetic. The peptide moiety can range in length from about 5 amino acids to about 40 amino acids. The peptide moiety may have structural modifications, such as to enhance stability or direct conformational properties. Any of the structural modifications described below may be used.

RGD peptide moieties for use in the compositions and methods of the invention may be linear or cyclic and may be modified, e.g., glycosylated or methylated, to facilitate targeting to specific tissues. good. RGD-containing peptides and peptidomimetics can use D-amino acids as well as synthetic RGD mimetics. In addition to RGD, other moieties that target integrin ligands can be used. Preferred conjugates of this ligand target PECAM-1 or VEGF.

A "cell-penetrating peptide" is capable of penetrating cells, eg, microbial cells, such as bacterial or fungal cells, or mammalian cells, such as human cells. Peptides that penetrate microbial cells include, for example, α-helical linear peptides (e.g. LL-37 or Ceropin P1), disulfide bond-containing peptides (e.g. α-defensins, β-defensins or bactenecins), or may be a peptide containing only one or two predominant amino acids (eg, PR-39 or indolicidin). Cell-penetrating peptides may also include nuclear localization signals (NLS). For example, the cell-penetrating peptide can be a dichotomous amphipathic peptide, such as MPG, derived from the fusion peptide domain of HIV-1 gp41 and the NLS of SV40 large T antigen (Simeoni et al., Nucl. Acids Res. 31 :2717-2724, 2003).

C. Carbohydrate Conjugates In certain embodiments of the compositions and methods of the invention, the iRNA oligonucleotide further comprises a carbohydrate. Carbohydrate-conjugated iRNAs, as described herein, are advantageous for the delivery of nucleic acids in vivo, and the compositions are suitable for in vivo therapeutic use. As used herein, a "carbohydrate" has at least 6 carbon atoms (which may be linear, branched or cyclic), with oxygen, nitrogen or sulfur at each carbon atom. A compound that is a carbohydrate itself, composed of one or more monosaccharide units to which atoms are attached; or has as part thereof a carbohydrate moiety composed of one or more monosaccharide units. , each of the monosaccharide units has at least 6 carbon atoms (which may be linear, branched or cyclic), to each carbon atom an oxygen, nitrogen or sulfur atom is attached. Refers to any of the compounds. Typical carbohydrates include sugars (monosaccharides, disaccharides, trisaccharides, and oligosaccharides containing about 4, 5, 6, 7, 8, or 9 monosaccharide units), as well as starches, glycogen, cellulose, and polysaccharide gums. Polysaccharides such as Particular monosaccharides include sugars of HBV or higher (e.g., HBV, C6, C7, or C8); disaccharides and trisaccharides include sugars of two or three monosaccharide units (e.g., HBV, C6, C7 or C8).

などのＮ－アセチルガラクトサミンである。 In one embodiment, carbohydrate conjugates for use in the compositions and methods of the invention are simple sugars. In one embodiment, the monosaccharide is

N-acetylgalactosamine, such as

からなる群から選択される。 In another embodiment, carbohydrate conjugates for use in the compositions and methods of the invention are

selected from the group consisting of.

が挙げられ、Ｘ又はＹの一方がオリゴヌクレオチドである場合、他方は水素である。 Other exemplary carbohydrate conjugates for use in embodiments described herein include, but are not limited to:

and when one of X or Y is an oligonucleotide, the other is hydrogen.

In certain embodiments, the carbohydrate conjugate further comprises one or more additional ligands as described above, such as, but not limited to, PK modulators and/or cell penetrating peptides.

Additional carbohydrate conjugates (and linkers) suitable for use in the present invention are disclosed in PCT publications WO 2014/179620 and WO 2014, each of which is incorporated by reference in its entirety. /179627 pamphlet is included.

D. Linkers In certain embodiments, the conjugates or ligands described herein can be attached to iRNA oligonucleotides using a variety of linkers, which can be cleavable or non-cleavable.

The term "linker" or "binding group" refers to an organic moiety that connects two parts of a compound, eg, covalently bonds two parts of a compound. Linkers typically include direct bonds or atoms such as oxygen or sulfur, units such as NR8, C(O), C(O)NH, SO, SO2, SO2NH, or substituted or unsubstituted Alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, arylalkyl, arylalkenyl, arylalkynyl, heteroarylalkyl, heteroarylalkenyl, heteroarylalkynyl, heterocyclylalkyl, heterocyclylalkenyl, heterocyclylalkynyl, aryl, heteroaryl , heterocyclyl, cycloalkyl, cycloalkenyl, alkylarylalkyl, alkylarylalkenyl, alkylarylalkynyl, alkenylarylalkyl, alkenylarylalkenyl, alkenylarylalkynyl, alkynylararylalkyl, alkynylarylalkenyl, alkynylarylalkynyl, alkylheteroarylalkyl, Alkylheteroarylalkenyl, alkylheteroarylalkynyl, alkenylheteroarylalkyl, alkenylheteroarylalkenyl, alkenylheteroarylalkynyl, alkynylheteroarylalkyl, alkynylheteroarylalkenyl, alkynylheteroarylalkynyl, alkylheterocyclylalkyl, alkylheterocyclylalkenyl, alkylheterocyclyl Alkynyl, alkenylheterocyclylalkyl, alkenylheterocyclylalkenyl, alkenylheterocyclylalkynyl, alkynylheterocyclylalkyl, alkynylheterocyclylalkenyl, alkynylheterocyclylalkynyl, alkylaryl, alkenylaryl, alkynylaryl, alkylheteroaryl, alkenylheteroaryl, alkynylheteroaryl (one or more Multiple methylenes interrupted by O, S, S(O), SO2, N(R8), C(O), substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocyclic or which may be terminated; where R8 is hydrogen, acyl, aliphatic or substituted aliphatic. In one embodiment, the linker has about 1-24 atoms, 2-24, 3-24, 4-24, 5-24, 6-24, 6-18, 7-18, 8-18 atoms. , 7-17, 8-17, 6-16, 7-16, or 8-16 atoms.

A cleavable linking group is one that is sufficiently stable outside the cell, but after entering the target cell, is cleaved to release the two moieties held together by the linker. In a preferred embodiment, the cleavable linking group is present in the target cell or under the first reference conditions (which may be selected, for example, to mimic or represent intracellular conditions), in the subject's blood, or in the second reference condition. at least about 10x, 20x, 30x, 40x, 50x, 60x, 70x, 80x, 90x under conditions (which may be selected to mimic or represent conditions found in blood or serum, for example). Cuts twice or more, or at least about 100 times faster.

Cleavable linking groups are sensitive to cleavage agents such as pH, redox potential or the presence of degradable molecules. Generally, cleaving agents are more prevalent or found at higher levels or activity inside cells compared to serum or blood. Examples of such degrading agents include, for example, reducing agents such as oxidizing or reductases present within the cell or mercaptans capable of degrading redox-cleavable bonding groups by reduction or redox agents without substrate specificity; esterases; agents capable of forming endosomes or acidic environments, e.g. agents that result in a pH below 5; acid-cleavable binding groups by acting as a general acid; Enzymes, peptidases (which may be substrate specific), and phosphatases that can hydrolyze or degrade

Cleavable linking groups such as disulfide bonds can be pH sensitive. While the pH of human serum is 7.4, the average intracellular pH is slightly lower, ranging from about 7.1 to 7.3. Endosomes have a more acidic pH in the range of 5.5-6.0, and lysosomes have an even more acidic pH of approximately 5.0. Some linkers will have a cleavable linking group that is cleaved at a preferred pH, thereby releasing the cationic lipid from the ligand inside the cell or into a desired compartment of the cell.

The linker may include a cleavable linking group that is cleavable by a particular enzyme. The type of cleavable linking group incorporated into the linker may depend on the targeted cell. For example, a liver-targeted ligand can be attached to a cationic lipid via a linker containing an ester group. Because hepatocytes are rich in esterases, this linker will be cleaved more efficiently in hepatocytes compared to cell types that are not rich in esterases. Other cell types rich in esterases include lung, renal cortex, and testicular cells.

Linkers containing peptide bonds can be used in targeting peptidase-rich cell types such as hepatocytes and synovial cells.

Generally, the suitability of a candidate cleavable binding group can be assessed by testing the ability of a decomposition agent (or decomposition condition) to cleave the candidate binding group. It may also be desirable to test the ability of candidate cleavable binding groups to resist cleavage upon contact with blood or other non-target tissues. Thus, the relative susceptibility to cleavage can be determined between a first condition and a second condition, where the first condition is selected to indicate cleavage within the target cell and the second condition is selected to exhibit cleavage within the target cell. is selected to indicate cleavage in other tissues or biological fluids, such as blood or serum. This evaluation can be performed in a cell-free system, intracellularly, in cell culture, in an organ or tissue culture, or in a whole animal. It may be useful to perform an initial evaluation in cell-free or culture conditions and confirm by further evaluation in whole animals. In a preferred embodiment, useful candidate compounds are intracellular (or selected to mimic intracellular conditions) compared to blood or serum (or in vitro conditions selected to mimic extracellular conditions). cleaved at least about 2, 4, 10, 20, 30, 40, 50, 60, 70, 80, 90, or about 100 times faster under in vitro conditions).

i. Redox-cleavable Linking Groups In one embodiment, the cleavable linking group is a redox-cleavable linking group that is cleaved after reduction or oxidation. An example of a reductively cleavable linking group is a disulfide linking group (-SS-). To determine whether a candidate cleavable binding group is a suitable "reductively cleavable binding group" or is suitable for use with a particular iRNA moiety and a particular targeting agent, for example, Attention may be drawn to the methods described herein. For example, candidates can be evaluated by incubation with dithiothreitol (DTT), or other reducing agents using reagents known in the art, which can be observed within cells, e.g. target cells. Mimics the speed of cutting. Candidates can also be evaluated under conditions selected to mimic blood or serum conditions. In one, the candidate compound is cleaved by about 10% or less in blood. In other embodiments, useful candidate compounds are intracellular (or mimic intracellular conditions) as compared to blood (or in vitro conditions selected to mimic extracellular conditions). at least about 2, 4, 10, 20, 30, 40, 50, 60, 70, 80, 90, or about 100 times faster under selected in vitro conditions). The cleavage rate of candidate compounds was determined using standard enzyme kinetic assays under conditions chosen to mimic the intracellular medium and compared to conditions chosen to mimic the extracellular medium. obtain.

ii. Phosphate-Based Cleavable Linking Groups In another embodiment, the cleavable linker comprises a phosphate-based cleavable linking group. Phosphate-based cleavable linking groups are cleaved by agents that degrade or hydrolyze phosphate groups. An example of an agent that cleaves phosphate groups within a cell is an enzyme such as an intracellular phosphatase. Examples of phosphate-based linking groups are -O-P(O)(ORk)-O-, -O-P(S)(ORk)-O-, -O-P(S)(SRk)- O-, -S-P(O)(ORk)-O-, -O-P(O)(ORk)-S-, -S-P(O)(ORk)-S-, -O-P( S)(ORk)-S-, -S-P(S)(ORk)-O-, -O-P(O)(Rk)-O-, -O-P(S)(Rk)-O- , -SP(O)(Rk)-O-, -SP(S)(Rk)-O-, -SP(O)(Rk)-S-, -OP(S) (Rk)-S-. Preferred embodiments include -O-P(O)(OH)-O-, -O-P(S)(OH)-O-, -O-P(S)(SH)-O-, -S- P(O)(OH)-O-, -O-P(O)(OH)-S-, -S-P(O)(OH)-S-, -O-P(S)(OH)- S-, -S-P(S)(OH)-O-, -O-P(O)(H)-O-, -O-P(S)(H)-O-, -S-P( O)(H)-O, -S-P(S)(H)-O-, -S-P(O)(H)-S-, -O-P(S)(H)-S- be. A preferred embodiment is -OP(O)(OH)-O-. These candidates can be evaluated using methods similar to those described above.

iii. Acid-cleavable linking group In another embodiment, the cleavable linker comprises an acid-cleavable linking group. Acid-cleavable linking groups are linking groups that are cleaved under acidic conditions. In preferred embodiments, the acid-cleavable linking group has a pH of about 6.5 or less (e.g., about 6.0, 5.75, 5.5, 5.25, 5.0, or less). It is cleaved in an acidic environment or by agents such as enzymes that can act as common acids. Within cells, certain low pH organelles such as endosomes and lysosomes can provide a cleavage environment for acid-cleavable linking groups. Examples of acid-cleavable linking groups include, but are not limited to, hydrazones, esters, and esters of amino acids. Acid-cleavable groups can be represented by the general formula -C=NN-, C(O)O, or -OC(O). A preferred embodiment is when the carbon bonded to the oxygen of the ester (alkoxy group) is an aryl group, a substituted alkyl group, or a tertiary alkyl group such as dimethylpentyl or t-butyl. These candidates can be evaluated using methods similar to those described above.

iv. Ester-Based Linking Groups In another embodiment, the cleavable linker comprises an ester-based cleavable linking group. Ester-based cleavable linking groups are cleaved by intracellular enzymes such as esterases and amylases. Examples of ester-based cleavable linking groups include, but are not limited to, alkylene, alkenylene, and esters of alkynylene groups. The ester-cleavable bonding group is represented by the general formula -C(O)O- or -OC(O)-. These candidates can be evaluated using methods similar to those described above.

v. Peptide-Based Cleavage Groups In yet another embodiment, the cleavable linker comprises a peptide-based cleavable linking group. Peptide-based cleavable linking groups are cleaved by intracellular enzymes such as peptidases and proteases. Peptide-based cleavable groups are peptide bonds formed between amino acids to give oligopeptides (eg, dipeptides, tripeptides, etc.) and polypeptides. Peptide-based cleavable groups do not include amide groups (-C(O)NH-). An amide group can be formed between any alkylene, alkenylene or alkynylene. Peptide bonds are a special type of amide bond that is formed between amino acids to give peptides and proteins. Peptide-based cleavage groups are generally limited to peptide bonds (ie, amide bonds) formed between amino acids to give peptides and proteins, and do not include the entire amide functionality. Peptide-based cleavable linking groups have the general formula -NHCHRAC(O)NHCHRBC(O)-, where RA and RB are the R groups of two adjacent amino acids. These candidates can be evaluated using methods similar to those described above.

が挙げられ、Ｘ又はＹの一方がオリゴヌクレオチドである場合、他方は水素である。 In one embodiment, the iRNA of the invention is conjugated to a carbohydrate via a linker. Non-limiting examples of iRNA carbohydrate conjugates with linkers of the compositions and methods of the invention include, but are not limited to:

and when one of X or Y is an oligonucleotide, the other is hydrogen.

In certain embodiments of the compositions and methods of the invention, the ligand is one or more "GalNAc" (N-acetylgalactosamine) derivatives attached via a divalent or trivalent branched linker. be.

のいずれかに示される構造の群から選択される二価又は三価の分枝鎖状リンカーにコンジュゲートされ、
式中：
ｑ２Ａ、ｑ２Ｂ、ｑ３Ａ、ｑ３Ｂ、ｑ４Ａ、ｑ４Ｂ、ｑ５Ａ、ｑ５Ｂ及びｑ５Ｃが、出現するごとに独立して、０～２０を表し、繰返し単位は、同一又は異なっていてもよく；
Ｐ２Ａ、Ｐ２Ｂ、Ｐ３Ａ、Ｐ３Ｂ、Ｐ４Ａ、Ｐ４Ｂ、Ｐ５Ａ、Ｐ５Ｂ、Ｐ５Ｃ、Ｔ２Ａ、Ｔ２Ｂ、Ｔ３Ａ、Ｔ３Ｂ、Ｔ４Ａ、Ｔ４Ｂ、Ｔ４Ａ、Ｔ５Ｂ、Ｔ５Ｃがそれぞれ、出現するごとに独立して、存在しないか、ＣＯ、ＮＨ、Ｏ、Ｓ、ＯＣ（Ｏ）、ＮＨＣ（Ｏ）、ＣＨ２、ＣＨ２ＮＨ又はＣＨ２Ｏであり；
Ｑ２Ａ、Ｑ２Ｂ、Ｑ３Ａ、Ｑ３Ｂ、Ｑ４Ａ、Ｑ４Ｂ、Ｑ５Ａ、Ｑ５Ｂ、Ｑ５Ｃが、出現するごとに独立して、存在しないか、アルキレン、置換アルキレンであり、ここで１つ又は複数のメチレンが、Ｏ、Ｓ、Ｓ（Ｏ）、ＳＯ２、Ｎ（ＲＮ）、Ｃ（Ｒ’）＝Ｃ（Ｒ”）、Ｃ≡Ｃ又はＣ（Ｏ）のうちの１つ又は複数により中断又は終端されてもよく；
Ｒ２Ａ、Ｒ２Ｂ、Ｒ３Ａ、Ｒ３Ｂ、Ｒ４Ａ、Ｒ４Ｂ、Ｒ５Ａ、Ｒ５Ｂ、Ｒ５Ｃがそれぞれ、出現するごとに独立して、存在しないか、ＮＨ、Ｏ、Ｓ、ＣＨ２、Ｃ（Ｏ）Ｏ、Ｃ（Ｏ）ＮＨ、ＮＨＣＨ（Ｒａ）Ｃ（Ｏ）、－Ｃ（Ｏ）－ＣＨ（Ｒａ）－ＮＨ－、ＣＯ、ＣＨ＝Ｎ－Ｏ、

又はヘテロシクリルであり；
Ｌ２Ａ、Ｌ２Ｂ、Ｌ３Ａ、Ｌ３Ｂ、Ｌ４Ａ、Ｌ４Ｂ、Ｌ５Ａ、Ｌ５Ｂ及びＬ５Ｃが、リガンドを表し；即ち、それぞれ、出現するごとに独立して、単糖（ＧａｌＮＡｃなど）、二糖、三糖、四糖、オリゴ糖、又は多糖であり；Ｒａが、Ｈ又はアミノ酸側鎖である。三価コンジュゲートＧａｌＮＡｃ誘導体は、ＲＮＡｉ剤とともに使用されて、式（ＸＸＸＶＩ）：

のものなどの標的遺伝子の発現を阻害するのに特に有用であり、
式中、Ｌ５Ａ、Ｌ５Ｂ及びＬ５Ｃが、ＧａｌＮＡｃ誘導体などの単糖を表す。 In one embodiment, the dsRNA of the invention has formulas (XXXII) to (XXXV):

conjugated to a divalent or trivalent branched linker selected from the group of structures shown in any of
During the ceremony:
Each occurrence of q2A, q2B, q3A, q3B, q4A, q4B, q5A, q5B and q5C independently represents 0 to 20, and the repeating units may be the same or different;
Each occurrence of P2A, P2B, P3A, P3B, P4A, P4B, P5A, P5B, P5C, T2A, T2B, T3A, T3B, T4A, T4B, T4A, T5B, T5C independently indicates whether they do not exist or CO, NH, O, S, OC(O), NHC(O), CH2, CH2NH or CH2O;
Each occurrence of Q2A, Q2B, Q3A, Q3B, Q4A, Q4B, Q5A, Q5B, Q5C is independently absent, alkylene, substituted alkylene, where one or more methylenes are O, may be interrupted or terminated by one or more of S, S(O), SO2, N(RN), C(R')=C(R''), C≡C or C(O);
Each occurrence of R2A, R2B, R3A, R3B, R4A, R4B, R5A, R5B, R5C independently indicates whether it does not exist or NH, O, S, CH2, C(O)O, C(O) NH, NHCH(Ra)C(O), -C(O)-CH(Ra)-NH-, CO, CH=N-O,

or heterocyclyl;
L2A, L2B, L3A, L3B, L4A, L4B, L5A, L5B and L5C represent the ligands; that is, each occurrence independently of a monosaccharide (such as GalNAc), a di-saccharide, a trisaccharide, a tetrasaccharide , oligosaccharide, or polysaccharide; Ra is H or an amino acid side chain. Trivalent conjugated GalNAc derivatives are used with RNAi agents to form formula (XXXVI):

It is particularly useful for inhibiting the expression of target genes such as those of
In the formula, L5A, L5B and L5C represent monosaccharides such as GalNAc derivatives.

Examples of suitable divalent and trivalent branched linking groups to be conjugated to GalNAc derivatives include, but are not limited to, the structures listed above as Formulas II, VII, XI, X, and XIII. It will be done.

Representative patents teaching the preparation of RNA conjugates include, but are not limited to, U.S. Pat. Nos. 4,828,979; 4,948,882; 5,218,105; No. 5,525,465; No. 5,541,313; No. 5,545,730; No. 5,552,538; No. 5,578,717 No. 5,580,731; No. 5,591,584; No. 5,109,124; No. 5,118,802; No. 5,138,045 No. 5,414,077; No. 5,486,603; No. 5,512,439; No. 5,578,718; No. 5,608,046 No. 4,587,044; No. 4,605,735; No. 4,667,025; No. 4,762,779; No. 4,789,737 No. 4,824,941; No. 4,835,263; No. 4,876,335; No. 4,904,582; No. 4,958,013 No. 5,082,830; No. 5,112,963; No. 5,214,136; No. 5,082,830; No. 5,112,963 No. 5,214,136; No. 5,245,022; No. 5,254,469; No. 5,258,506; No. 5,262,536 No. 5,272,250; No. 5,292,873; No. 5,317,098; No. 5,371,241, No. 5,391,723 No. 5,416,203, No. 5,451,463; No. 5,510,475; No. 5,512,667; No. 5,514,785 No. 5,565,552; No. 5,567,810; No. 5,574,142; No. 5,585,481; No. 5,587,371 5,595,726; 5,597,696; 5,599,923; 5,599,928 and 5,688,941 No. 6,294,664; No. 6,320,017; No. 6,576,752; No. 6,783,931; No. 6,900,297 No. 7,037,646; and No. 8,106,022, the entire contents of each of which are incorporated herein by reference.

It is not necessary that all positions of a given compound be uniformly modified; in fact, two or more of the above modifications may be combined into a single nucleoside in a single compound or even within an iRNA. can be incorporated into. The invention also includes iRNA compounds that are chimeric compounds.

In the context of the present invention, a "chimeric" iRNA compound or "chimera" is an iRNA compound that comprises two or more chemically distinct regions, each consisting of at least one monomer unit, i.e., a nucleotide in the case of a dsRNA compound. , preferably dsRNA. These iRNAs typically include at least one region in which the RNA is configured to confer increased resistance to nuclease degradation, increased cellular uptake, and/or increased binding affinity for a target nucleic acid. Qualified as . Additional regions of the iRNA can serve as substrates for enzymes capable of cleaving RNA:DNA or RNA:RNA hybrids. As an example, RNase H is a cellular endonuclease that cleaves the RNA strand of an RNA:DNA duplex. Activation of RNase H therefore results in cleavage of the RNA target, thereby greatly increasing the efficiency of iRNA inhibition of gene expression. As a result, when chimeric dsRNAs are used, comparable results are often obtained with shorter iRNAs compared to phosphorothioate deoxy dsRNAs that hybridize to the same target region. Cleavage of an RNA target can routinely be detected by gel electrophoresis and, optionally, by related nucleic acid hybridization techniques known in the art.

In some cases, the RNA of an iRNA can be modified with a non-ligand group. Several non-ligand molecules have been conjugated to iRNA to improve its activity, cellular distribution or uptake, and procedures for performing such conjugations are available in the scientific literature. Such non-ligand moieties include cholesterol (Kubo, T. et al., Biochem. Biophys. Res. Comm., 2007, 365(1):54-61; Letsinger et al., Proc. Natl. Acad. USA, 1989, 86:6553), cholic acid (Manoharan et al., Bioorg. Med. Chem. Lett., 1994, 4:1053), thioethers such as hexyl-S-tritylthiol (Manoharan et al., Ann. N. Y. Acad. Sci., 1992, 660: 306; Manoharan et al., Bioorg. Med. Chem. Let., 1993, 3: 2765), thiocholesterol (Oberhauser et al., Nucl. Acids Res ., 1992, 20:533), aliphatic chains, such as dodecanediol or undecyl residues (Saison-Behmoaras et al., EMBO J., 1991, 10:111; Kabanov et al., FEBS Lett., 1990, 259:327; Svinarchuk et al., Biochimie, 1993, 75:49), phospholipids such as di-hexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H -phosphonates (Manoharan et al., Tetrahedron Lett., 1995, 36:3651; Shea et al., Nucl. Acids Res., 1990, 18:3777), polyamines or polyethylene glycol chains (Manoharan et al., N ucleosides & nucleotides, 1995 , 14:969), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36:3651), palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264:229), or octa decylamine or a lipid moiety such as a hexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277:923). Representative US patents teaching the preparation of such RNA conjugates are listed above. A typical conjugation protocol involves the synthesis of RNA with an amino linker at one or more positions of the sequence. The amino group is then reacted with the conjugated molecule using a suitable coupling or activating reagent. The conjugation reaction can be performed with the RNA still bound to a solid support or after cleavage of the RNA in solution phase. Purification of RNA conjugates by HPLC typically yields pure conjugates.

V. Delivery of iRNA of the invention to cells in a subject, such as a human subject (e.g., a subject in need of an iRNA agent, such as a subject with a disease, disorder, or condition associated with HBV infection). Delivery of iRNA can be accomplished in a number of different ways. For example, delivery can be performed by contacting a cell with an iRNA of the invention either in vitro or in vivo. In vivo delivery can also be performed directly by administering to a subject a composition comprising the iRNA, eg, dsRNA. Alternatively, in vivo delivery can be accomplished indirectly by administering one or more vectors encoding and directing the expression of the iRNA. These alternatives are discussed further below.

In general, any method of delivering nucleic acid molecules (in vitro or in vivo) can be adapted for use with the iRNAs of the invention (see, e.g., Akhtar S. and Julian, hereby incorporated by reference in their entirety). RL., (1992) Trends Cell. Biol. 2(5): 139-144 and WO 94/02595 pamphlet). For in vivo delivery, factors considered for delivering an iRNA molecule include, for example, biological stability of the delivered molecule, prevention of non-specific effects, and accumulation of the delivered molecule in the target tissue. Can be mentioned. Non-specific effects of iRNA can be minimized by local administration, eg, by direct injection or implantation into tissue, or by administering the formulation locally. Local administration to the treatment site maximizes the local concentration of the agent, limits the exposure of the agent to systemic tissues that may be adversely affected by or degrade the agent, and reduces the total dose of iRNA molecules administered. can be reduced. Several studies have shown successful knockdown of gene products when iRNA is administered locally. For example, intraocular delivery of VEGF dsRNA by intravitreal injection in cynomolgus monkeys (Tolentino, MJ. et al., (2004) Retina 24:132-138) and subretinal injection in mice (Reich, SJ. et al. (2003)). Mol.Vis.9:210-216) were both shown to prevent neovascularization in experimental models of age-related macular degeneration. Furthermore, direct intratumoral administration of dsRNA in mice can reduce tumor volume (Pille, J. et al. (2005) Mol. Ther. 11:267-274) and prolong survival of tumor-bearing mice ( Kim, WJ. et al., (2006) Mol. Ther. 14:343-350; Li, S. et al., (2007) Mol. Ther. 15:515-523). RNA interference can be delivered locally to the central nervous system by direct injection (Dorn, G. et al., (2004) Nucleic Acids 32:e49; Tan, PH. et al. (2005) Gene Ther. 12:59-66 Makimura, H. et al. (2002) BMC Neurosci. 3:18; Shishkina, GT., et al. (2004) Neuroscience 129:521-528; Thakker, ER., et al. (2004) Proc.N atl Acad.Sci.U.S.A. 101:17270-17275; Akaneya, Y., et al. (2005) J. Neurophysiol. 93:594-602) and local delivery to the lungs by intranasal administration (Howard , KA. et al., (2006) Mol. Ther. 14:476-484; Zhang, X. et al., (2004) J. Biol. Chem. 279: 10677-10684; Bitko, V. et al. , (2005) Nat. Med. 11:50-55). For systemic administration of iRNA for the treatment of diseases, the RNA can be modified or delivered using drug delivery systems; both methods involve rapid production of dsRNA by endonucleases and exonucleases in vivo. plays a role in preventing decomposition. Modification of the RNA or pharmaceutical carrier can also allow targeting of the iRNA composition to target tissues and avoid undesirable off-target effects. iRNA molecules can be modified by chemical attachment to lipophilic groups, such as cholesterol, to improve cellular uptake and prevent degradation. For example, iRNA against ApoB conjugated to a lipophilic cholesterol moiety was administered systemically to mice and knockdown of apoB mRNA was obtained in both liver and jejunum (Soutschek, J. et al., (2004) Nature 432 :173-178). Conjugation of iRNA to aptamers has been shown to inhibit tumor growth and mediate tumor regression in mouse models of prostate cancer (McNamara, JO. et al., (2006) Nat. Biotechnol. 24: 1005-1015). In alternative embodiments, iRNA can be delivered using drug delivery systems such as nanoparticles, dendrimers, polymers, liposomes, or cationic delivery systems. The positively charged cationic delivery system facilitates binding of iRNA molecules (which are negatively charged) and also improves interactions at the negatively charged cell membrane, allowing efficient uptake of iRNA by cells. do. Cationic lipids, dendrimers, or polymers can be attached to the iRNA or in vesicles or micelles that enclose the iRNA (e.g., Kim SH. et al., (2008) Journal of Controlled Release 129(2): 107-116 ) can be induced to form. Vesicle or micelle formation further prevents iRNA degradation when administered systemically. Methods for making and administering cationic iRNA complexes are well within the ability of those skilled in the art (e.g., Sorensen, DR., et al., incorporated herein by reference in their entirety). 2003) J. Mol. Biol 327:761-766; Verma, UN. et al., (2003) Clin. Cancer Res. 9:1291-1300; Arnold, AS et al., (2007) J. Hypertens. 25 :197-205). Some non-limiting examples of drug delivery systems useful for systemic delivery of iRNA include DOTAP (Sorensen, DR., et al. (2003), supra; Verma, UN. et al., (2003); (see above), Oligofectamine, "solid nucleic acid lipid particles" (Zimmermann, TS. et al., (2006) Nature 441:111-114), cardiolipin (Chien, PY. et al., (2005) Cancer Gene Ther. 12:321-328; Pal, A. et al., (2005) Int J. Oncol. 26:1087-1091), polyethyleneimine (Bonnet ME. et al., (2008) Pharm. Res. Aug 16 Epub ahead of print; Aigner, A. (2006) J. Biomed. Biotechnol. 71659), Arg-Gly-Asp (RGD) peptide (Liu, S. (2006) Mol. Pharm. 3:472-487), and polyamide amine (Tomalia, DA. et al., (2007) Biochem. Soc. Trans. 35:61-67; Yoo, H. et al., (1999) Pharm. Res. 16:1799-1804). In certain embodiments, the iRNA is complexed with cyclodextrin for systemic administration. Methods for administration and pharmaceutical compositions of iRNAs and cyclodextrins can be found in US Pat. No. 7,427,605, which is incorporated herein by reference in its entirety.

A. iRNA of the invention encoded by a vector
iRNAs targeting HBV genes can be expressed from transcription units inserted into DNA or RNA vectors (eg, Couture, A, et al., TIG. (1996), 12:5-10; Skillern, A. et al., International PCT Publication No. WO 00/22113, Conrad, International PCT Publication No. WO 00/22114, and Conrad, US Pat. No. 6,054,299). Expression can be transient (on the order of hours to weeks) or sustained (from weeks to months or more) depending on the particular construct used and the target tissue or cell type. These transgenes can be introduced as linear constructs, circular plasmids, or viral vectors, which can be integrating or non-integrating vectors. Transgenes can also be constructed to allow them to be inherited as extrachromosomal plasmids (Gassmann, et al., Proc. Natl. Acad. Sci. USA (1995) 92:1292).

Individual strands of an iRNA can be transcribed from a promoter in an expression vector. If two separate chains are expressed, eg, to produce dsRNA, the two separate expression vectors can be co-introduced into the target cell (eg, by transfection or infection). Alternatively, each individual strand of dsRNA can be transcribed by a promoter located on the same expression plasmid. In one embodiment, the dsRNA is expressed as an inverted repeat polynucleotide joined by a linker polynucleotide sequence so that it has a stem-loop structure.

iRNA expression vectors are generally DNA plasmids or viral vectors. Expression vectors compatible with eukaryotic cells, preferably vertebrate cells, can be used to produce recombinant constructs for expression of the iRNAs described herein. Eukaryotic expression vectors are well known in the art and are available from many commercial sources. Typically, such vectors are provided containing convenient restriction sites for insertion of the desired nucleic acid segment. Delivery of the iRNA expression vector can be, for example, by intravenous or intramuscular administration, by administration to target cells transplanted from the patient and then reintroduced into the patient, or to the desired target cells. Systemic delivery may be by any other means.

iRNA expression plasmids can be transfected into target cells as complexes with cationic lipid carriers (eg, Oligofectamine) or non-cationic lipid-based carriers (eg, Transit-TKO™). Multiple transfections of lipids for iRNA-mediated knockdown targeting different regions of the target RNA over a period of one week or more are also envisioned by the present invention. Successful introduction of a vector into a host cell can be monitored using a variety of known methods. For example, transient transfection can be demonstrated using a reporter such as a fluorescent marker such as green fluorescent protein (GFP). Stable transfection of cells ex vivo can be ensured using markers that confer resistance to certain environmental factors (eg, antibiotics and drugs) on the transfected cells, such as hygromycin B resistance.

Viral vector systems that may be used with the methods and compositions described herein include, but are not limited to, (a) adenoviral vectors; (b) lentiviral vectors, Moloney murine leukemia virus, and the like. (c) adeno-associated virus vectors; (d) herpes simplex virus vectors; (e) SV40 vectors; (f) polyomavirus vectors; (g) papillomavirus vectors; (h) picornavirus vectors. (i) orthopox, such as vaccinia virus vectors or avian pox, such as canarypox or fowlpox, and (j) helper-dependent or attenuated adenoviruses. Replication-defective viruses may also be advantageous. Different vectors may or may not integrate into the genome of the cell. The construct may optionally include viral sequences for transfection. Alternatively, the constructs can be incorporated into vectors capable of episomal replication, such as EPV and EBV vectors. Constructs for recombinant expression of iRNA generally require regulatory elements, such as promoters, enhancers, etc., to ensure expression of the iRNA within the target cell. Other aspects contemplated for vectors and constructs are discussed further below.

Vectors useful for delivery of iRNA will contain sufficient regulatory elements (promoter, enhancer, etc.) for expression of the iRNA in the desired target cell or tissue. Regulatory elements can be selected to provide either constitutive expression or regulated/inducible expression.

Expression of iRNAs can be precisely regulated, for example, by using inducible regulatory sequences that are sensitive to specific physiological regulators, such as blood glucose levels, or hormones (Docherty et al., 1994, FASEB J. 8:20-24). Such inducible expression systems suitable for controlling the expression of dsRNA in cells or mammals include, for example, ecdysone, estrogen, progesterone, tetracycline, chemical inducers of dimerization, and isopropyl-β-D1-thiogalactopyrano. (IPTG). One skilled in the art will be able to select appropriate regulatory/promoter sequences based on the intended use of the iRNA transgene.

Viral vectors containing nucleic acid sequences encoding iRNA can be used. For example, retroviral vectors can be used (see Miller et al., Meth. Enzymol. 217:581-599 (1993)). These retroviral vectors contain the necessary components for proper packaging and integration of the viral genome into host cell DNA. Nucleic acid sequences encoding iRNAs are cloned into one or more vectors that facilitate delivery of the nucleic acids to patients. Further details about retroviral vectors can be found, for example, in Boesen et al. , Biotherapy 6:291-302 (1994), which describes the use of retroviral vectors to deliver the mdr1 gene to hematopoietic stem cells to make them more resistant to chemotherapy. has been done. Other references demonstrating the use of retroviral vectors in gene therapy include Clowes et al. , J. Clin. Invest. 93:644-651 (1994); Kiem et al. , Blood 83:1467-1473 (1994); Salmons and Gunzberg, Human Gene Therapy 4:129-141 (1993); and Grossman and Wilson, Curr. Open. in Genetics and Devel. 3:110-114 (1993). Lentiviral vectors contemplated for use include, for example, U.S. Pat. No. 6,143,520; U.S. Pat. No. 5,665,557; 5,981,276.

Adenoviruses are also contemplated for use in delivering the iRNAs of the invention. Adenoviruses, for example, are particularly attractive vehicles for delivering genes to respiratory epithelia. Adenoviruses primarily infect respiratory epithelia and cause mild disease. Other targets for adenovirus-based delivery systems are the liver, central nervous system, endothelial cells, and muscle. Adenoviruses have the advantage of being able to infect non-dividing cells. Kozarsky and Wilson, Current Opinion in Genetics and Development 3:499-503 (1993) provides an overview of adenovirus-based gene therapy. Bout et al. , Human Gene Therapy 5:3-10 (1994) demonstrated the use of adenoviral vectors to transfer genes to the respiratory epithelium of rhesus monkeys. Other examples of the use of adenoviruses in gene therapy include Rosenfeld et al. , Science 252:431-434 (1991); Rosenfeld et al. , Cell 68:143-155; Mastrangeli et al. (1992), J. Clin. Invest. 91:225-234 (1993); PCT Publication WO 94/12649 pamphlet; and Wang et al. , Gene Therapy 2:775-783 (1995). AV vectors suitable for expressing the iRNAs featured in the present invention, methods for constructing recombinant AV vectors, and methods for delivering the vectors into target cells are described by Xia H et al. (2002), Nat. Biotech. 20:1006-1010.

Adeno-associated virus (AAV) vectors can also be used to deliver the iRNAs of the invention (Walsh et al., Proc. Soc. Exp. Biol. Med. 204:289-300 (1993); U.S. Pat. , 436, 146). In one embodiment, the iRNA can be expressed as two separate complementary single-stranded RNA molecules from a recombinant AAV vector with either a U6 or H1 RNA promoter, or a cytomegalovirus (CMV) promoter, for example. . AAV vectors suitable for expressing the dsRNAs featured in this invention, methods for constructing recombinant AV vectors, and methods for delivering the vectors into target cells are disclosed herein in their entirety by reference. Samulski R et al. (1987), J. Virol. 61:3096-3101; Fisher KJ et al. (1996), J. Virol, 70:520-532; Samulski R et al. (1989), J. Virol. 63:3822-3826; US Patent No. 5,252,479; US Patent No. 5,139,941; International Patent Application No. WO 94/13788 pamphlet; and International Patent Application No. WO It is described in pamphlet No. 93/24641.

Another viral vector suitable for delivery of the iRNA of the invention is a vaccinia virus, e.g. attenuated vaccinia such as Modified Virus Ankara (MVA) or NYVAC, avian pox such as fowlpox or canarypox. It's a pox virus.

The tropism of viral vectors can be modified by pseudotyping the vector with envelope proteins or other surface antigens from other viruses, or by optionally substituting different viral capsid proteins. obtain. For example, lentiviral vectors can be pseudotyped using surface proteins from vesicular stomatitis virus (VSV), rabies, Ebola, Mokola, etc. AAV vectors can be made to target different cells by engineering the vectors to express different capsid protein serotypes. See, for example, Rabinowitz J E et al., the entire disclosure of which is incorporated herein by reference. (2002), J Virol 76:791-801.

Pharmaceutical formulations of vectors can include the vector in an acceptable diluent or can include a sustained release matrix in which the gene delivery vehicle is embedded. Alternatively, if a complete gene delivery vector, such as a retroviral vector, can be produced intact from recombinant cells, the pharmaceutical formulation can include one or more cells that produce the gene delivery system.

VI. Pharmaceutical Compositions of the Invention The invention also includes pharmaceutical compositions and formulations comprising the iRNA of the invention. Also provided herein, in one embodiment, is a pharmaceutical composition containing an iRNA described herein and a pharmaceutically acceptable carrier. Pharmaceutical compositions containing iRNA are useful for treating diseases or disorders associated with HBV gene expression or activity. Such pharmaceutical compositions are formulated based on the mode of delivery. One example is a composition formulated for systemic administration via parenteral administration, eg, subcutaneous (SC) or intravenous (IV) delivery. Another example is a composition formulated for direct delivery to the brain parenchyma, eg, by injection into the brain, such as by continuous pump infusion. Pharmaceutical compositions of the invention may be administered in a dosage sufficient to inhibit HBV gene expression.

In one embodiment, an iRNA agent of the invention is administered to a subject as a weight-based dose. A "weight-based dose" (eg, a dose in mg/kg) is a dose of an iRNA agent that can vary depending on the weight of the subject. In another embodiment, the iRNA agent is administered to the subject as a fixed dose. A "fixed dose" (eg, a dose in mg) means that one dose of the iRNA agent is used for all subjects, regardless of any specific subject-related factors, such as body weight. In one particular embodiment, the fixed dose of an iRNA agent of the invention is based on a given weight or age.

Generally, a suitable dose of an iRNA of the invention may range from about 0.001 to about 200.0 milligrams per kilogram of body weight of the recipient per day, generally from about 1 to 50 mg per kilogram of body weight per day of the recipient. . For example, dsRNA can be administered at about 0.01 mg/kg, about 0.05 mg/kg, about 0.5 mg/kg, about 1 mg/kg, about 1.5 mg/kg, about 2 mg/kg, about 3 mg/kg per single dose. kg, about 10 mg/kg, about 20 mg/kg, about 30 mg/kg, about 40 mg/kg, or about 50 mg/kg.

For example, dsRNA may be about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1. 2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2. 5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3. 8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9, 9. 1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, or about 10 mg/kg. Values and ranges intermediate to those recited are also intended to be part of the invention.

In another embodiment, the dsRNA is about 0.1 to about 50 mg/kg, about 0.25 to about 50 mg/kg, about 0.5 to about 50 mg/kg, about 0.75 to about 50 mg/kg, about 1 to about 50 mg/kg, about 1.5 to about 50 mg/kg, about 2 to about 50 mg/kg, about 2.5 to about 50 mg/kg, about 3 to about 50 mg/kg, about 3.5 to about 50 mg /kg, about 4 to about 50 mg/kg, about 4.5 to about 50 mg/kg, about 5 to about 50 mg/kg, about 7.5 to about 50 mg/kg, about 10 to about 50 mg/kg, about 15 to about about 50 mg/kg, about 20 to about 50 mg/kg, about 20 to about 50 mg/kg, about 25 to about 50 mg/kg, about 25 to about 50 mg/kg, about 30 to about 50 mg/kg, about 35 to about 50 mg /kg, about 40 to about 50 mg/kg, about 45 to about 50 mg/kg, about 0.1 to about 45 mg/kg, about 0.25 to about 45 mg/kg, about 0.5 to about 45 mg/kg, about 0.75 to about 45 mg/kg, about 1 to about 45 mg/kg, about 1.5 to about 45 mg/kg, about 2 to about 45 mg/kg, about 2.5 to about 45 mg/kg, about 3 to about 45 mg /kg, about 3.5 to about 45 mg/kg, about 4 to about 45 mg/kg, about 4.5 to about 45 mg/kg, about 5 to about 45 mg/kg, about 7.5 to about 45 mg/kg, about 10 to about 45 mg/kg, about 15 to about 45 mg/kg, about 20 to about 45 mg/kg, about 20 to about 45 mg/kg, about 25 to about 45 mg/kg, about 25 to about 45 mg/kg, about 30 to about About 45 mg/kg, about 35 to about 45 mg/kg, about 40 to about 45 mg/kg, about 0.1 to about 40 mg/kg, about 0.25 to about 40 mg/kg, about 0.5 to about 40 mg/kg , about 0.75 to about 40 mg/kg, about 1 to about 40 mg/kg, about 1.5 to about 40 mg/kg, about 2 to about 40 mg/kg, about 2.5 to about 40 mg/kg, about 3 to about About 40 mg/kg, about 3.5 to about 40 mg/kg, about 4 to about 40 mg/kg, about 4.5 to about 40 mg/kg, about 5 to about 40 mg/kg, about 7.5 to about 40 mg/kg , about 10 to about 40 mg/kg, about 15 to about 40 mg/kg, about 20 to about 40 mg/kg, about 20 to about 40 mg/kg, about 25 to about 40 mg/kg, about 25 to about 40 mg/kg, about 30 to about 40 mg/kg, about 35 to about 40 mg/kg, about 0.1 to about 30 mg/kg, about 0.25 to about 30 mg/kg, about 0.5 to about 30 mg/kg, about 0.75 to about about 30 mg/kg, about 1 to about 30 mg/kg, about 1.5 to about 30 mg/kg, about 2 to about 30 mg/kg, about 2.5 to about 30 mg/kg, about 3 to about 30 mg/kg, about 3.5 to about 30 mg/kg, about 4 to about 30 mg/kg, about 4.5 to about 30 mg/kg, about 5 to about 30 mg/kg, about 7.5 to about 30 mg/kg, about 10 to about 30 mg /kg, about 15 to about 30 mg/kg, about 20 to about 30 mg/kg, about 20 to about 30 mg/kg, about 25 to about 30 mg/kg, about 0.1 to about 20 mg/kg, about 0.25 to about About 20 mg/kg, about 0.5 to about 20 mg/kg, about 0.75 to about 20 mg/kg, about 1 to about 20 mg/kg, about 1.5 to about 20 mg/kg, about 2 to about 20 mg/kg , about 2.5 to about 20 mg/kg, about 3 to about 20 mg/kg, about 3.5 to about 20 mg/kg, about 4 to about 20 mg/kg, about 4.5 to about 20 mg/kg, about 5 to about Administered at a dose of about 20 mg/kg, about 7.5 to about 20 mg/kg, about 10 to about 20 mg/kg, or about 15 to about 20 mg/kg. Values and ranges intermediate to those recited are also intended to be part of the invention.

For example, dsRNA is about 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4. 2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5. 5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6. 8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9, 9.1, 9.2, 9.3, 9.4, It may be administered at a dose of 9.5, 9.6, 9.7, 9.8, 9.9, or about 10 mg/kg. Values and ranges intermediate to those recited are also intended to be part of the invention.

In another embodiment, the dsRNA is about 0.5 to about 50 mg/kg, about 0.75 to about 50 mg/kg, about 1 to about 50 mg/kg, about 1.5 to about 50 mg/kg, about 2 to About 50 mg/kg, about 2.5 to about 50 mg/kg, about 3 to about 50 mg/kg, about 3.5 to about 50 mg/kg, about 4 to about 50 mg/kg, about 4.5 to about 50 mg/kg , about 5 to about 50 mg/kg, about 7.5 to about 50 mg/kg, about 10 to about 50 mg/kg, about 15 to about 50 mg/kg, about 20 to about 50 mg/kg, about 20 to about 50 mg/kg , about 25 to about 50 mg/kg, about 25 to about 50 mg/kg, about 30 to about 50 mg/kg, about 35 to about 50 mg/kg, about 40 to about 50 mg/kg, about 45 to about 50 mg/kg, about 0.5 to about 45 mg/kg, about 0.75 to about 45 mg/kg, about 1 to about 45 mg/kg, about 1.5 to about 45 mg/kg, about 2 to about 45 mg/kg, about 2.5 to about about 45 mg/kg, about 3 to about 45 mg/kg, about 3.5 to about 45 mg/kg, about 4 to about 45 mg/kg, about 4.5 to about 45 mg/kg, about 5 to about 45 mg/kg, about 7.5 to about 45 mg/kg, about 10 to about 45 mg/kg, about 15 to about 45 mg/kg, about 20 to about 45 mg/kg, about 20 to about 45 mg/kg, about 25 to about 45 mg/kg, about 25 to about 45 mg/kg, about 30 to about 45 mg/kg, about 35 to about 45 mg/kg, about 40 to about 45 mg/kg, about 0.5 to about 40 mg/kg, about 0.75 to about 40 mg/kg , about 1 to about 40 mg/kg, about 1.5 to about 40 mg/kg, about 2 to about 40 mg/kg, about 2.5 to about 40 mg/kg, about 3 to about 40 mg/kg, about 3.5 to about about 40 mg/kg, about 4 to about 40 mg/kg, about 4.5 to about 40 mg/kg, about 5 to about 40 mg/kg, about 7.5 to about 40 mg/kg, about 10 to about 40 mg/kg, about 15 to about 40 mg/kg, about 20 to about 40 mg/kg, about 20 to about 40 mg/kg, about 25 to about 40 mg/kg, about 25 to about 40 mg/kg, about 30 to about 40 mg/kg, about 35 to about About 40 mg/kg, about 0.5 to about 30 mg/kg, about 0.75 to about 30 mg/kg, about 1 to about 30 mg/kg, about 1.5 to about 30 mg/kg, about 2 to about 30 mg/kg , about 2.5 to about 30 mg/kg, about 3 to about 30 mg/kg, about 3.5 to about 30 mg/kg, about 4 to about 30 mg/kg, about 4.5 to about 30 mg/kg, about 5 to about About 30 mg/kg, about 7.5 to about 30 mg/kg, about 10 to about 30 mg/kg, about 15 to about 30 mg/kg, about 20 to about 30 mg/kg, about 20 to about 30 mg/kg, about 25 to about About 30 mg/kg, about 0.5 to about 20 mg/kg, about 0.75 to about 20 mg/kg, about 1 to about 20 mg/kg, about 1.5 to about 20 mg/kg, about 2 to about 20 mg/kg , about 2.5 to about 20 mg/kg, about 3 to about 20 mg/kg, about 3.5 to about 20 mg/kg, about 4 to about 20 mg/kg, about 4.5 to about 20 mg/kg, about 5 to about Administered at a dose of about 20 mg/kg, about 7.5 to about 20 mg/kg, about 10 to about 20 mg/kg, or about 15 to about 20 mg/kg. In one embodiment, dsRNA is administered at a dose of about 10 mg/kg to about 30 mg/kg. Values and ranges intermediate to those recited are also intended to be part of the invention.

For example, targets include approximately 0.1, 0.125, 0.15, 0.175, 0.2, 0.225, 0.25, 0.275, 0.3, 0.325, 0.35 , 0.375, 0.4, 0.425, 0.45, 0.475, 0.5, 0.525, 0.55, 0.575, 0.6, 0.625, 0.65, 0 .675, 0.7, 0.725, 0.75, 0.775, 0.8, 0.825, 0.85, 0.875, 0.9, 0.925, 0.95, 0.975 , 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2 .3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3 .6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4 .9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2 , 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5 , 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8 , 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10, 10.5, 11 , 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19, 19 .5, 20, 20.5, 21, 21.5, 22, 22.5, 23, 23.5, 24, 24.5, 25, 25.5, 26, 26.5, 27, 27.5 , 28, 28.5, 29, 29.5, 30, 31, 32, 33, 34, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47 , 48, 49, or about 50 mg/kg, can be administered, for example, subcutaneously or intravenously. Values and ranges intermediate to those recited are also intended to be part of the invention.

In some embodiments, the subject receives a dose of about 0.1, 0.125, 0.15, 0.175, 0.2, 0.225, 0.25, 0.275, 0.3, 0 .325, 0.35, 0.375, 0.4, 0.425, 0.45, 0.475, 0.5, 0.525, 0.55, 0.575, 0.6, 0.625 , 0.65, 0.675, 0.7, 0.725, 0.75, 0.775, 0.8, 0.825, 0.85, 0.875, 0.9, 0.925, 0 .95, 0.975, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1 , 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4 , 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7 , 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6 .1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7 .4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8 .7, 8.8, 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10 , 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18 .5, 19, 19.5, 20, 20.5, 21, 21.5, 22, 22.5, 23, 23.5, 24, 24.5, 25, 25.5, 26, 26.5 , 27, 27.5, 28, 28.5, 29, 29.5, 30, 31, 32, 33, 34, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44 , 45, 46, 47, 48, 49, or about 50 mg/kg, for example, administered subcutaneously or intravenously. A multiple dose regimen can include administering a therapeutic amount of iRNA daily, such as for 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, or more.

In other embodiments, the subject is administered a dose of about 0.1, 0.125, 0.15, 0.175, 0.2, 0.225, 0.25, 0.275, 0.3, 0. 325, 0.35, 0.375, 0.4, 0.425, 0.45, 0.475, 0.5, 0.525, 0.55, 0.575, 0.6, 0.625, 0.65, 0.675, 0.7, 0.725, 0.75, 0.775, 0.8, 0.825, 0.85, 0.875, 0.9, 0.925, 0. 95, 0.975, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6. 1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7. 4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8. 7, 8.8, 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18. 5, 19, 19.5, 20, 20.5, 21, 21.5, 22, 22.5, 23, 23.5, 24, 24.5, 25, 25.5, 26, 26.5, 27, 27.5, 28, 28.5, 29, 29.5, 30, 31, 32, 33, 34, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, Multiple doses of a therapeutic amount of iRNA, such as 45, 46, 47, 48, 49, or about 50 mg/kg, are administered, eg, subcutaneously or intravenously. Repeated dose regimens can include regular administration of a therapeutic amount of iRNA, such as every other day, every second day, every third day, twice a week, once a week, every other week, or once a month.

In certain embodiments, for example, when a composition of the invention comprises dsRNA and a lipid as described herein, the subject receives from about 0.01 mg/kg to about 5 mg/kg, about 0. 01 mg/kg to about 10 mg/kg, about 0.05 mg/kg to about 5 mg/kg, about 0.05 mg/kg to about 10 mg/kg, about 0.1 mg/kg to about 5 mg/kg, about 0.1 mg/kg kg to about 10 mg/kg, about 0.2 mg/kg to about 5 mg/kg, about 0.2 mg/kg to about 10 mg/kg, about 0.3 mg/kg to about 5 mg/kg, about 0.3 mg/kg to About 10 mg/kg, about 0.4 mg/kg to about 5 mg/kg, about 0.4 mg/kg to about 10 mg/kg, about 0.5 mg/kg to about 5 mg/kg, about 0.5 mg/kg to about 10 mg /kg, about 1 mg/kg to about 5 mg/kg, about 1 mg/kg to about 10 mg/kg, about 1.5 mg/kg to about 5 mg/kg, about 1.5 mg/kg to about 10 mg/kg, about 2 mg/kg kg to about 2.5 mg/kg, about 2 mg/kg to about 10 mg/kg, about 3 mg/kg to about 5 mg/kg, about 3 mg/kg to about 10 mg/kg, about 3.5 mg/kg to about 5 mg/kg kg, about 4 mg/kg to about 5 mg/kg, about 4.5 mg/kg to about 5 mg/kg, about 4 mg/kg to about 10 mg/kg, about 4.5 mg/kg to about 10 mg/kg, about 5 mg/kg ~about 10mg/kg, about 5.5mg/kg to about 10mg/kg, about 6mg/kg to about 10mg/kg, about 6.5mg/kg to about 10mg/kg, about 7mg/kg to about 10mg/kg, about 7.5 mg/kg to about 10 mg/kg, about 8 mg/kg to about 10 mg/kg, about 8.5 mg/kg to about 10 mg/kg, about 9 mg/kg to about 10 mg/kg, or about 9.5 mg/kg A therapeutic amount of iRNA can be administered, such as from kg to about 10 mg/kg. Values and ranges intermediate to those recited are also intended to be part of the invention.

For example, dsRNA may be about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1. 2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2. 5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3. 8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9, 9. 1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, or about 10 mg/kg. Values and ranges intermediate to those recited are also intended to be part of the invention.

In certain embodiments of the invention, for example, a double-stranded RNAi agent has a modification (e.g., one or more motifs of three identical modifications to three consecutive nucleotides), e.g., at or near the cleavage site of the agent. When containing one such motif, six phosphorothioate linkages, and a ligand, such agent may be present at about 0.01 to about 0.5 mg/kg, about 0.01 to about 0.4 mg/kg, about 0.01 to about 0.3 mg/kg, about 0.01 to about 0.2 mg/kg, about 0.01 to about 0.1 mg/kg, about 0.01 mg/kg to about 0.09 mg/kg, about 0.01 mg/kg - about 0.08 mg/kg, about 0.01 mg/kg - about 0.07 mg/kg, about 0.01 mg/kg - about 0.06 mg/kg, about 0.01 mg/kg - about 0.05 mg/kg, About 0.02 to about 0.5 mg/kg, about 0.02 to about 0.4 mg/kg, about 0.02 to about 0.3 mg/kg, about 0.02 to about 0.2 mg/kg, about 0 .02 to about 0.1 mg/kg, about 0.02 mg/kg to about 0.09 mg/kg, about 0.02 mg/kg to about 0.08 mg/kg, about 0.02 mg/kg to about 0.07 mg/kg kg, about 0.02 mg/kg to about 0.06 mg/kg, about 0.02 mg/kg to about 0.05 mg/kg, about 0.03 to about 0.5 mg/kg, about 0.03 to about 0. 4 mg/kg, about 0.03 to about 0.3 mg/kg, about 0.03 to about 0.2 mg/kg, about 0.03 to about 0.1 mg/kg, about 0.03 mg/kg to about 0. 09mg/kg, about 0.03mg/kg to about 0.08mg/kg, about 0.03mg/kg to about 0.07mg/kg, about 0.03mg/kg to about 0.06mg/kg, about 0.03mg /kg to about 0.05 mg/kg, about 0.04 to about 0.5 mg/kg, about 0.04 to about 0.4 mg/kg, about 0.04 to about 0.3 mg/kg, about 0.04 ～about 0.2mg/kg, about 0.04～about 0.1mg/kg, about 0.04mg/kg～about 0.09mg/kg, about 0.04mg/kg～about 0.08mg/kg, about 0 .04 mg/kg to about 0.07 mg/kg, about 0.04 mg/kg to about 0.06 mg/kg, about 0.05 to about 0.5 mg/kg, about 0.05 to about 0.4 mg/kg, about 0.05 to about 0.3 mg/kg, about 0.05 to about 0.2 mg/kg, about 0.05 to about 0.1 mg/kg, about 0.05 mg/kg to about 0.09 mg/kg, Administered at a dose of about 0.05 mg/kg to about 0.08 mg/kg, or about 0.05 mg/kg to about 0.07 mg/kg. Values and ranges intermediate to the foregoing recited values are also intended to be part of the invention; for example, the RNAi agent may be administered to a subject at a dose of about 0.015 mg/kg to about 0.45 mg/kg. can be done.

For example, the RNAi agent, e.g., the RNAi agent in the pharmaceutical composition, can be used at about 0.01 mg/kg, 0.0125 mg/kg, 0.015 mg/kg, 0.0175 mg/kg, 0.02 mg/kg, 0.0225 mg/kg. kg, 0.025mg/kg, 0.0275mg/kg, 0.03mg/kg, 0.0325mg/kg, 0.035mg/kg, 0.0375mg/kg, 0.04mg/kg, 0.0425mg/kg, 0.045mg/kg, 0.0475mg/kg, 0.05mg/kg, 0.0525mg/kg, 0.055mg/kg, 0.0575mg/kg, 0.06mg/kg, 0.0625mg/kg, 0. 065mg/kg, 0.0675mg/kg, 0.07mg/kg, 0.0725mg/kg, 0.075mg/kg, 0.0775mg/kg, 0.08mg/kg, 0.0825mg/kg, 0.085mg/ kg, 0.0875mg/kg, 0.09mg/kg, 0.0925mg/kg, 0.095mg/kg, 0.0975mg/kg, 0.1mg/kg, 0.125mg/kg, 0.15mg/kg, 0.175mg/kg, 0.2mg/kg, 0.225mg/kg, 0.25mg/kg, 0.275mg/kg, 0.3mg/kg, 0.325mg/kg, 0.35mg/kg, 0. It may be administered at a dose of 375 mg/kg, 0.4 mg/kg, 0.425 mg/kg, 0.45 mg/kg, 0.475 mg/kg, or about 0.5 mg/kg. Values intermediate to those listed above are also intended to be part of this invention.

In some embodiments, the RNAi agent is about 100 mg to about 900 mg, such as about 100 mg to about 850 mg, about 100 mg to about 800 mg, about 100 mg to about 750 mg, about 100 mg to about 700 mg, about 100 mg to about 650 mg, about 100mg to about 600mg, about 100mg to about 550mg, about 100mg to about 500mg, about 200mg to about 850mg, about 200mg to about 800mg, about 200mg to about 750mg, about 200mg to about 700mg, about 200mg to about 650mg, about 200mg to about about 600 mg, about 200 mg to about 550 mg, about 200 mg to about 500 mg, about 300 mg to about 850 mg, about 300 mg to about 800 mg, about 300 mg to about 750 mg, about 300 mg to about 700 mg, about 300 mg to about 650 mg, about 300 mg to about 600 mg , about 300 mg to about 550 mg, about 300 mg to about 500 mg, about 400 mg to about 850 mg, about 400 mg to about 800 mg, about 400 mg to about 750 mg, about 400 mg to about 700 mg, about 400 mg to about 650 mg, about 400 mg to about 600 mg, about Administered as a fixed dose of 400 mg to about 550 mg, or between about 400 mg and about 500 mg.

In some embodiments, the RNAi agent is about 100 mg, about 125 mg, about 150 mg, about 175 mg, 200 mg, about 225 mg, about 250 mg, about 275 mg, about 300 mg, about 325 mg, about 350 mg, about 375 mg, about 400 mg, about 425mg, about 450mg, about 475mg, about 500mg, about 525mg, about 550mg, about 575mg, about 600mg, about 625mg, about 650mg, about 675mg, about 700mg, about 725mg, about 750mg, about 775mg, about 800mg, about 825mg, Administered as a fixed dose of about 850 mg, about 875 mg, or about 900 mg.

The pharmaceutical composition may be administered for 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, and 21, 22, 23, 24 minutes, or about 25 minutes. It may be administered by intravenous infusion over a period of time such as minutes. Administration can be repeated periodically, such as weekly, biweekly (ie, every two weeks) for a month, two months, three months, four months, or more. After the initial treatment regimen, therapeutic agents may be administered less frequently. For example, after weekly or biweekly administration for three months, administration can be repeated once a month for six months or a year or more.

The pharmaceutical composition can be administered once a day, or the iRNA can be administered as two, three or more sub-doses at appropriate intervals throughout the day, or even via continuous infusion or sustained release formulation. The administration may also be administered using controlled delivery. In that case, the iRNA contained in each subdose would need to be correspondingly smaller in order to achieve the total daily dose. Dosage units may also be formulated for delivery over several days, eg, using conventional sustained release formulations that provide sustained release of iRNA over a period of several days. Sustained release formulations are well known in the art and can be used with the agents of the present invention as they are particularly useful for delivering drugs to specific sites. In this embodiment, the dosage unit contains a corresponding multiple of the daily dose.

In other embodiments, a single administration of the pharmaceutical composition can be long-lasting such that subsequent doses are administered no more than 3, 4, or 5 days apart, or no more than 1, 2, 3, or 4 weeks apart. Administered at intervals. In certain embodiments of the invention, a single dose of a pharmaceutical composition of the invention is administered once a week. In other embodiments of the invention, a single dose of a pharmaceutical composition of the invention is administered bi-monthly. In some embodiments of the invention, a single dose of a pharmaceutical composition of the invention is administered monthly, bimonthly, or quarterly (ie, every three months).

Those skilled in the art will appreciate that certain factors will effectively treat a subject, including, but not limited to, the severity of the disease or condition, previous treatments, the subject's general health and/or age, and other illnesses present. It will be appreciated that this may affect the dosage and time required for treatment. Additionally, treatment of a subject with a therapeutically effective amount of a composition can include a single treatment or a series of treatments. Effective dosages and in vivo half-lives for individual iRNAs encompassed by the present invention can be determined using conventional methodologies or using appropriate animal models as described elsewhere herein. An approximate estimate can be made based on in vivo studies conducted.

The pharmaceutical compositions of the invention may be administered in several ways, depending on whether local or systemic treatment is required and the area to be treated. Administration may be local (e.g. by transdermal patch), pulmonary by inhalation or insufflation of a powder or aerosol, e.g. by a nebulizer; intratracheal, intranasal, epidermal and transdermal, oral or parenteral administration. obtain. Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; subcutaneous administration, e.g. by an implanted device; or intracranial administration, e.g. by intraparenchymal, intrathecal or intraventricular administration. It will be done.

iRNA can be delivered to target specific tissues, such as the liver (eg, hepatocytes of the liver).

Pharmaceutical compositions and formulations for topical administration include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, solutions and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickening agents and the like may be necessary or desirable. Covered condoms, gloves, etc. may also be useful. Suitable topical formulations include those in which the iRNA that characterizes the invention is in admixture with topical delivery agents such as lipids, liposomes, fatty acids, fatty acid esters, steroids, chelating agents, and surfactants. Suitable lipids and liposomes include neutral (e.g. dioleyl phosphatidyl DOPE ethanolamine, dimyristoyl phosphatidylcholine DMPC, distearoyl phosphatidylcholine), anionic (e.g. dimyristoyl phosphatidylglycerol DMPG) and cationic (e.g. dioleyl phosphatidyl choline) tetramethylaminopropyl DOTAP and dioleyl phosphatidylethanolamine DOTMA). The iRNAs that characterize the invention can be encapsulated in liposomes or can be complexed to liposomes, particularly cationic liposomes. Alternatively, the iRNA may be complexed to a lipid, particularly a cationic lipid. Suitable fatty acids and esters include, but are not limited to, arachidonic acid, oleic acid, eicosanoic acid, lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate. , monoolein, dilaurin, glyceryl 1-monocaprate, 1-dodecyl azacycloheptan-2-one, acylcarnitine, acylcholine, or C1-20 alkyl ester (e.g., isopropyl myristate IPM), monoglyceride, diglyceride, or pharmaceuticals thereof (including legally acceptable salts). Topical formulations are described in detail in US Pat. No. 6,747,014, which is incorporated herein by reference.

A. iRNA Formulations Comprising Membrane Molecular Assemblies The iRNA for use in the compositions and methods of the invention can be formulated for delivery in membrane molecular assemblies, such as liposomes or micelles. As used herein, the term "liposome" refers to a vesicle composed of amphiphilic lipids arranged in at least one bilayer, e.g., a bilayer or multiple bilayers. . Liposomes include unilamellar and multilamellar vesicles with membranes formed from lipophilic materials and an aqueous interior. The aqueous portion contains the iRNA composition. The lipophilic material separates the aqueous interior from the aqueous exterior and typically does not contain the iRNA composition, but in some cases may. Liposomes are useful for transport and delivery of active ingredients to the site of action. Liposome membranes are structurally similar to biological membranes, so when liposomes are attached to tissue, the liposome bilayer fuses with the cell membrane bilayer. As liposome and cell fusion proceeds, the internal aqueous contents containing iRNA are delivered to the cell, where the iRNA can specifically bind to and mediate the target RNA. In some cases, the liposomes are also specifically targeted, eg, to direct the iRNA to a particular cell type.

Liposomes containing iRNA agents can be prepared by a variety of methods. In one example, the lipid component of the liposome is dissolved in a detergent such that micelles are formed with the lipid component. For example, the lipid component can be an amphipathic cationic lipid or a lipid conjugate. Detergents can have high critical micelle concentrations and can be non-ionic. Exemplary detergents include cholate, CHAPS, octylglucoside, deoxycholate, and lauroylsarcosine. The iRNA agent preparation is then added to the micelles containing the lipid component. Cationic groups in the lipids interact with and condense around the iRNA agent to form liposomes. After condensation, the detergent is removed, eg, by dialysis, resulting in a liposomal formulation of the iRNA agent.

If necessary, carrier compounds that assist the condensation can be added during the condensation reaction, for example by controlled addition. For example, the carrier compound can be a polymer other than a nucleic acid, such as spermine or spermidine. pH may also be adjusted to aid condensation.

Methods for producing stable polynucleotide delivery vehicles that incorporate polynucleotide/cationic lipid complexes as components of the delivery vehicle are described, for example, in WO 96/37194, the entire contents of which are incorporated herein by reference. Further information is provided in the issue pamphlet. Liposome formation is described by Felgner, P. L. et al. , Proc. Natl. Acad. Sci. USA 8:7413-7417, 1987; US Pat. No. 4,897,355; US Pat. No. 5,171,678; Bangham et al. ,M. Mol. Biol. 23:238, 1965; Olson et al. , Biochim. Biophys. Acta 557:9, 1979; Szoka et al. , Proc. Natl. Acad. Sci. 75:4194, 1978; Mayhew et al. , Biochim. Biophys. Acta 775:169, 1984; Kim et al. , Biochim. Biophys. Acta 728:339, 1983; and Fukunaga et al. , Endocrinol. 115:757, 1984. Commonly used techniques for preparing appropriately sized lipid aggregates for use as delivery vehicles include sonication and freeze-thaw and extrusion (eg, Mayer et al., Biochim. Biophys. Acta 858:161, 1986). If consistently small (50-200 nm) and relatively uniform aggregates are desired, microfluidization may be used (Mayhew et al., Biochim. Biophys. Acta 775:169, 1984). These methods are easily adapted to package preparations of iRNA agents into liposomes.

Liposomes are divided into two major classes. Cationic liposomes are positively charged liposomes that interact with negatively charged nucleic acid molecules to form stable complexes. The positively charged nucleic acid/liposome complex binds to the negatively charged cell surface and is translocated into endosomes. The acidic pH within the endosome causes the liposomes to rupture, releasing their contents into the cytoplasm (Wang et al., Biochem. Biophys. Res. Commun., 1987, 147, 980-985).

pH-sensitive and negatively charged liposomes entrap nucleic acids rather than complexing with them. Both nucleic acids and lipids are similarly charged, so repulsion rather than complex formation occurs. Nevertheless, some nucleic acids are entrapped within the aqueous interior of these liposomes. pH-sensitive liposomes have been used to deliver nucleic acids encoding thymidine kinase genes to cell monolayers in culture. Expression of foreign genes was detected in target cells (Zhou et al., Journal of Controlled Release, 1992, 19, 269-274).

One major type of liposome composition contains phospholipids other than naturally occurring phosphatidylcholine. For example, neutral liposome compositions can be formed from dimyristoyl phosphatidylcholine (DMPC) or dipalmitoylphosphatidylcholine (DPPC). Anionic liposome compositions are generally formed from dimyristoyl phosphatidylglycerol, while anionic membranous liposomes are formed primarily from dioleyl phosphatidylethanolamine (DOPE). Other types of liposome compositions are formed from phosphatidylcholine (PC), such as, for example, soybean PC, and egg PC. Other types are formed from mixtures of phospholipids and/or phosphatidylcholines and/or cholesterol.

Examples of other methods for introducing liposomes into cells in vitro and in vivo include U.S. Pat. No. 5,283,185; U.S. Pat. No. 5,171,678; WO 94/ 00569 pamphlet; WO 93/24640 pamphlet; WO 91/16024 pamphlet; Felgner, J. Biol. Chem. 269:2550, 1994; Nabel, Proc. Natl. Acad. Sci. 90:11307, 1993; Nabel, Human Gene Ther. 3:649, 1992; Gershon, Biochem. 32:7143, 1993; and Strauss, EMBO J. 11:417, 1992.

Nonionic liposomal systems, particularly those containing nonionic surfactants and cholesterol, have also been tested to determine their utility in delivering drugs to the skin. Non-ionic liposome formulations containing Novasome™ I (glyceryl dilaurate/cholesterol/polyoxyethylene-10-stearyl ether) and Novasome™ II (glyceryl distearate/cholesterol/polyoxyethylene-10-stearyl ether) was used to deliver cyclosporin-A to the dermis of mouse skin. Results showed that such non-ionic liposome systems are effective in promoting the deposition of cyclosporin-A into different layers of the skin (Hu et al. S.T.P. Pharma. Sci. , 1994, 4(6) 466).

Liposomes also include "sterically stabilized" liposomes, which term as used herein refers to liposomes that include one or more specialized lipids that are incorporated into the liposome. When used, they result in enhanced circulation longevity compared to liposomes lacking such specialized lipids. Examples of sterically stabilized liposomes are those in which a portion of the vesicle-forming lipid portion of the liposome comprises (A) one or more glycolipids, such as monosialoganglioside GM1, or (B) polyethylene glycol ( derivatized with one or more hydrophilic polymers, such as PEG) moieties. Without being bound to any particular theory, it is known in the art, at least with respect to sterically stabilized liposomes containing gangliosides, sphingomyelins, or PEG-derivatized lipids, that these sterically stabilized The enhanced circulating half-life of modified liposomes is thought to result from reduced intracellular uptake of the reticuloendothelial system (RES) (Allen et al., FEBS Letters, 1987, 223, 42; Wu et al., Cancer Research, 1993, 53, 3765).

A variety of liposomes containing one or more glycolipids are known in the art. Papahadjopoulos et al. (Ann. NY Acad. Sci., 1987, 507, 64) reported the ability of monosialoganglioside GM1, galactocerebroside sulfate, and phosphatidylinositol to improve the blood half-life of liposomes. These findings were reported by Gabizon et al. (Proc. Natl. Acad. Sci. USA, 1988, 85, 6949). Both Allen et al. U.S. Pat. No. 4,837,028 and WO 88/04924, issued to the US Pat. . US Pat. No. 5,543,152 (Webb et al.) discloses liposomes containing sphingomyelin. Liposomes containing 1,2-sn-dimyristoylphosphatidylcholine are disclosed in WO 97/13499 (Lim et al.).

In one embodiment, cationic liposomes are used. Cationic liposomes have the advantage of being able to fuse to cell membranes. Although non-cationic liposomes cannot fuse as efficiently with cell membranes, they can be taken up by macrophages in vivo and used to deliver iRNA agents to macrophages.

Additional advantages of liposomes include: Liposomes derived from natural phospholipids are biocompatible and can be biodegradable; liposomes can incorporate a wide range of water- and lipid-soluble drugs. Liposomes can protect iRNA agents encapsulated in their internal compartments from metabolism and degradation (Rosoff, in “Pharmaceutical Dosage Forms,” Lieberman, Rieger and Banker (Eds.), 1988, volume 1, p.245). Important considerations in the preparation of liposome formulations are lipid surface charge, vesicle size and aqueous volume of the liposomes.

N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA), a positively charged synthetic cationic lipid, is used to spontaneously interact with nucleic acids. can act to form small liposomes that fuse with negatively charged lipids of the plasma membrane of tissue culture cells to form lipid-nucleic acid complexes that can result in the delivery of iRNA agents (e.g., Felgner et al. , P. L. et al., Proc. Natl. Acad. Sci. USA 8:7413-7417, 1987, and U.S. Pat. No. 4,897,355 for a description of DOTMA and its use with DNA. reference).

The DOTMA analog, 1,2-bis(oleyloxy)-3-(trimethylammonia)propane (DOTAP), can be used in combination with phospholipids to form DNA complex vesicles. Lipofectin™ Bethesda Research Laboratories, Gaithersburg, Md. ) is an effective method for delivering highly anionic nucleic acids into biological tissue culture cells containing positively charged DOTMA liposomes that spontaneously interact with negatively charged polynucleotides to form complexes. It is a drug. If sufficiently positively charged liposomes are used, the net charge of the resulting complex is also positive. The positively charged complexes thus prepared spontaneously attach to negatively charged cell surfaces, fuse with the cell membrane, and efficiently deliver functional nucleic acids into, for example, tissue culture cells. Another commercially available cationic lipid, 1,2-bis(oleyloxy)-3,3-(trimethylammonia)propane (“DOTAP”) (Boehringer Mannheim, Indianapolis, Indiana), has an ether linkage in the oleoyl moiety. It differs from DOTMA in that it is linked by an ester rather than by an ester.

Other reported cationic lipid compounds include 5-carboxyspermylglycine dioctaoleoylamide (“DOGS”) (Transfectam™), which is conjugated to one of two types of lipids; Promega, Madison, Wisconsin) and dipalmitoylphosphatidylethanolamine 5-carboxyspermyl-amide ("DPPES") conjugated to various moieties, including carboxyspermine (e.g. , U.S. Pat. No. 5,171,678).

Another cationic lipid conjugate involves derivatization of lipids with cholesterol formulated into liposomes (“DC-Chol”) in combination with DOPE (Gao, X. and Huang, L., Biochim. Biophys. Res. .Commun. 179:280, 1991). Lipopolylysine made by conjugating polylysine to DOPE has been reported to be effective for transfection in the presence of serum (Zhou, X. et al., Biochim. Biophys. Acta 1065:8, 1991). For certain cell lines, these liposomes containing conjugated cationic lipids are said to exhibit lower toxicity and provide more efficient transfection than DOTMA-containing compositions. Other commercially available cationic lipid products include DMRIE and DMRIE-HP (Vical, La Jolla, California) and Lipofectamine (DOSPA) (Life Technology, Inc., Gaithersburg, Maryland). Other cationic lipids suitable for the delivery of oligonucleotides are described in WO 98/39359 and WO 96/37194.

Liposomal formulations are particularly suited for topical administration, and liposomes exhibit several advantages over other formulations. Such advantages include reduced side effects associated with high systemic absorption of the administered drug, increased accumulation of the administered drug at the desired target, and the ability to administer iRNA agents into the skin. In some implementations, liposomes are used to deliver the iRNA agent to epidermal cells and to facilitate penetration of the iRNA agent into dermal tissues, such as the skin. For example, liposomes can be applied topically. Topical delivery to the skin of drugs formulated as liposomes has been reported (e.g. Weiner et al., Journal of Drug Targeting, 1992, vol. 2, 405-410 and du Plessis et al., Antiviral Research, 18, 1992: 259-265; Mannino, R. J. and Fould-Fogerite, S., Biotechniques 6: 682-690, 1988; Itani, T. et al., Gene 56: 267-276, 1987; Nicola u, C. et al. (1987) Meth. Enz. 149:157-176, 1987; Straubinger, R. M. and Papahadjopoulos, D. Meth. Enz. 101:512-527, 1983; Wang, C. Y. and See Huang, L., Proc. Natl. Acad. Sci. USA 84:7851-7855, 1987).

Nonionic liposomal systems, particularly those containing nonionic surfactants and cholesterol, have also been investigated to determine their utility in delivering drugs to the skin. Non-ionic liposome formulations containing Novasome I (glyceryl dilaurate/cholesterol/polyoxyethylene-10-stearyl ether) and Novasome II (glyceryl distearate/cholesterol/polyoxyethylene-10-stearyl ether) were used in the dermis of mouse skin. was used to deliver drugs. Such formulations containing iRNA agents are useful for treating skin diseases.

Liposomes containing iRNA can be made highly deformable. Such deformability may allow liposomes to penetrate pores that are smaller than the average radius of the liposome. For example, a transfersome is a type of deformable liposome. Transfersomes can be created by adding surface edge activators, usually surfactants, to standard liposome compositions. Transfersomes containing RiNA agents can be delivered, for example, by subcutaneous infection, to deliver iRNA agents to keratinocytes of the skin. To traverse intact mammalian skin, lipid vesicles must pass through a series of micropores, each with a diameter of less than 50 nm, under the influence of a suitable transdermal gradient. Moreover, because of their lipid properties, these transferosomes can be self-optimizing (e.g., adaptable to the shape of the pore), self-repairing, and often reach their targets without fragmentation. In many cases, it may be self-loading.

Other formulations suitable for the invention are described, for example, in WO 2008/042973, the entire contents of which are incorporated herein by reference.

Transfersomes are yet another type of liposome, highly deformable lipid aggregates that are attractive candidates as drug delivery vehicles. Transfersomes can also be described as lipid droplets, which are highly deformable and can therefore easily pass through pores smaller than droplets. Transfersomes are adaptable to the environment in which they are used, e.g. they are self-optimal (adapting to the shape of pores within the skin), self-repairing, and often reach their targets without fragmentation. reachable and often self-loading. To create transfersomes, surface edge activators, usually surfactants, can be added to standard liposome compositions. Transfersomes have been used to deliver serum albumin to the skin. Transfersome-mediated delivery of serum albumin has been shown to be as effective as subcutaneous injection of solutions containing serum albumin.

Surfactants find wide use in formulations such as emulsions (including microemulsions) and liposomes. The most common method of classifying and ranking the many different types of surfactants, both natural and synthetic, is through the use of hydrophilic/lipophilic balance (HLB). The nature of the hydrophilic group (also known as the "head") provides the most useful means of categorizing different surfactants used in formulations (Rieger, "Pharmaceutical Dosage Forms", Marcel Dekker, Inc. , New York, N.Y., 1988, p. 285).

If the surfactant molecules are not ionized, the surfactant is classified as a nonionic surfactant. Nonionic surfactants find wide application in pharmaceutical and cosmetic products and can be used over a wide range of pH values. Generally, their HLB values range from 2 to about 18 depending on their structure. Nonionic surfactants include nonionic esters such as ethylene glycol esters, propylene glycol esters, glyceryl esters, polyglyceryl esters, sorbitan esters, sucrose esters, and ethoxylated esters. Also included in this class are nonionic alkanolamides and ethers such as fatty alcohol ethoxylates, propoxylated alcohols, and ethoxylated/propoxylated block polymers. Polyoxyethylene surfactants are the most popular members of the nonionic surfactant class.

If a surfactant molecule carries a negative charge when dissolved or dispersed in water, the surfactant is classified as anionic. Anionic surfactants include carboxylates such as soaps, acyl lactylates, acylamides of amino acids, sulfuric esters such as alkyl sulfates and ethoxylated alkyl sulfates, sulfonates such as alkylbenzene sulfonates, acyl isethionates, and acyl tau. esters and sulfosuccinates, as well as phosphates. The most important members of the anionic surfactant class are alkyl sulfates and soaps.

If a surfactant molecule carries a positive charge when dissolved or dispersed in water, the surfactant is classified as cationic. Cationic surfactants include quaternary ammonium salts and ethoxylated amines. Quaternary ammonium salts are the most used members of this class.

If a surfactant molecule has the ability to carry either a positive or negative charge, the surfactant is classified as amphoteric. Amphoteric surfactants include acrylic acid derivatives, substituted alkylamides, N-alkyl betaines and phosphatides.

The use of surfactants in drug products, formulations and emulsions has been reviewed (Rieger, "Pharmaceutical Dosage Forms", Marcel Dekker, Inc., New York, NY, 1988, p. 285).

iRNA for use in the methods of the invention can also be provided as a micellar formulation. "Micelles" are a specific type of amphiphilic molecules arranged in a spherical configuration such that all the hydrophobic parts of the molecules face inward, leaving the hydrophilic parts in contact with the surrounding aqueous phase. Defined herein as a molecular assembly of. The opposite configuration exists if the environment is hydrophobic.

Mixed micellar formulations suitable for transdermal delivery can be prepared by mixing an aqueous solution of the siRNA composition, an alkali metal C8-C22 alkyl sulfate, and a micelle-forming compound. Exemplary micelle-forming compounds include lecithin, hyaluronic acid, pharmaceutically acceptable salts of hyaluronic acid, glycolic acid, lactic acid, chamomile extract, cucumber extract, oleic acid, linoleic acid, linolenic acid, monoolein, Monooleate, monolaurate, borage oil, evening primrose oil, menthol, trihydroxyoxocolanylglycine and its pharmaceutically acceptable salts, glycerin, polyglycerin, lysine, polylysine, triolein, polyoxyethylene ether and its analogs , polidocanol alkyl ethers and their analogs, chenodeoxycholates, deoxycholates, and mixtures thereof. The micelle-forming compound may be added simultaneously with or after the addition of the alkali metal alkyl sulfate. Mixed micelles are formed with virtually any type of mixing of the components, but with vigorous mixing to provide micelles of smaller size.

In one method, a first micelle composition containing an siRNA composition and at least an alkali metal alkyl sulfate is prepared. The first micelle composition is then mixed with at least three micelle-forming compounds to form a mixed micelle composition. In another method, the micelle composition is prepared by mixing at least one of the siRNA composition, the alkali metal alkyl sulfate, and the micelle-forming compound, followed by adding the remaining micelle-forming compound with vigorous mixing. .

Phenol and/or m-cresol may be added to the mixed micellar composition to stabilize the formulation and protect against bacterial growth. Alternatively, phenol and/or m-cresol may be added along with the micelle-forming component. Isotonic agents such as glycerin may also be added after formation of the mixed micelle composition.

For delivery of a micellar formulation as a spray, the formulation can be placed in an aerosol dispenser and the dispenser is filled with a propellant. The propellant under pressure is in liquid form in the dispenser. The proportions of the components are adjusted such that the aqueous phase and the propellant phase are one, ie, one phase is present. If two phases are present, the dispenser needs to be shaken before dispensing a portion of the contents, for example by a metering valve. The drug dose is delivered in a fine spray through a metered-dose valve.

Propellants may include hydrogen-containing chlorofluorocarbons, hydrogen-containing fluorocarbons, dimethyl ether and diethyl ether. In certain embodiments, HFA 134a (1,1,1,2 tetrafluoroethane) may be used.

Specific concentrations of essential components can be determined by relatively simple experimentation. For absorption via the oral cavity, it is often desirable to increase the dose, eg, at least two or three times, for injection or administration via the gastrointestinal tract.

B. Lipid Particles The iRNA, ie, dsRNA, of the invention may be completely encapsulated in a lipid formulation, eg, LNP, or may form other nucleic acid-lipid particles.

The term "LNP" as used herein refers to a stable nucleic acid-lipid particle. LNPs typically include cationic lipids, non-cationic lipids, and lipids that prevent particle aggregation (eg, PEG-lipid conjugates). LNPs have an extended circulation life after intravenous (i.v.) injection and accumulate at distal sites (e.g., sites physically separated from the site of administration), making them extremely useful for systemic applications. It is. LNPs include "pSPLPs," which include encapsulated condensing agent-nucleic acid complexes, as shown in PCT Publication No. WO 00/03683. Particles of the invention typically have a particle size of about 50 nm to about 150 nm, more typically about 60 nm to about 130 nm, more typically about 70 nm to about 110 nm, most typically about 70 nm to about 90 nm. average particle size and is substantially non-toxic. Additionally, the nucleic acids, when present in the nucleic acid-lipid particles of the invention, are resistant to degradation by nucleases in aqueous solutions. Nucleic acid-lipid particles and methods for their preparation are described, for example, in U.S. Pat. No. 5,976,567; U.S. Pat. No. 5,981,501; U.S. Pat. No. 6,534,484; Disclosed in Patent No. 6,586,410; U.S. Patent No. 6,815,432; U.S. Patent Application Publication No. 2010/0324120 and PCT Publication WO 96/40964 pamphlet. .

In one embodiment, the lipid to drug ratio (mass/mass ratio) (e.g., lipid to dsRNA ratio) is about 1:1 to about 50:1, about 1:1 to about 25:1, about 3: 1 to about 15:1, about 4:1 to about 10:1, about 5:1 to about 9:1, or about 6:1 to about 9:1. Ranges intermediate to the above ranges are also considered to be part of the invention.

Cationic lipids include, for example, N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N-(I-(2 ,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP), N-(I-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride ( DOTMA), N,N-dimethyl-2,3-dioleyloxy)propylamine (DODMA), 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 1,2-dilinolenyl Oxy-N,N-dimethylaminopropane (DLenDMA), 1,2-dilinoleylcarbamoyloxy-3-dimethylaminopropane (DLin-C-DAP), 1,2-dilinoley oxy-3-(dimethyl Amino) acetoxypropane (DLin-DAC), 1,2-dilinoleyoxy-3-morpholinopropane (DLin-MA), 1,2-dilinoleoyl-3-dimethylaminopropane (DLinDAP), 1,2-dilinoleylthio -3-dimethylaminopropane (DLin-S-DMA), 1-linoleoyl-2-linoleyloxy-3-dimethylaminopropane (DLin-2-DMAP), 1,2-dilinoleyloxy-3-trimethylamino Propane chloride salt (DLin-TMA.Cl), 1,2-dilinoleoyl-3-trimethylaminopropane chloride salt (DLin-TAP.Cl), 1,2-dilinoleyloxy-3-(N-methylpiperazino)propane ( DLin-MPZ), or 3-(N,N-dilinoleylamino)-1,2-propanediol (DLinAP), 3-(N,N-dioleylamino)-1,2-propanediol (DOAP) , 1,2-dilinoleyloxo-3-(2-N,N-dimethylamino)ethoxypropane (DLin-EG-DMA), 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA) ), 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA) or its analog, (3aR,5s,6aS)-N,N-dimethyl-2,2 -di((9Z,12Z)-octadeca-9,12-dienyl)tetrahydro-3aH-cyclopenta[d][1,3]dioxol-5-amine (ALN100), (6Z,9Z,28Z,31Z)-hepta Triaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate (MC3), 1,1'-(2-(4-(2-((2-(bis(2- hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethylazanediyl)didodecan-2-ol (Tech G1), or mixtures thereof. Cationic lipids can comprise about 20 mol% to about 50 mol%, or about 40 mol% of the total lipids present in the particle.

In another embodiment, the compound 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane can be used to prepare lipid-siRNA nanoparticles. The synthesis of 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane is described in U.S. Provisional Patent Application No. 61/2008, filed October 23, 2008, which is hereby incorporated by reference. No. 107,998.

In one embodiment, the lipid-siRNA particles include 40% 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane: 10% DSPC: 40% cholesterol: 10% PEG-C. - Contains DOMG (mol percent) with a particle size of 63.0±20 nm and a siRNA/lipid ratio of 0.027.

Ionic/non-cationic lipids include, but are not limited to, distearoylphosphatidylcholine (DSPC), dioleylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG) , dioleyl-phosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE), dioleyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE) -mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl-phosphatidyl-ethanolamine (DSPE), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1 - Can be anionic or neutral lipids, including trans-PE, 1-stearoyl-2-oleoyl-phosphatidy ethanolamine (SOPE), cholesterol, or mixtures thereof. Non-cationic lipids, if cholesterol is included, may represent about 5 mol% to about 90 mol%, about 10 mol%, or about 58 mol% of the total lipids present in the particle.

Conjugate lipids that inhibit particle aggregation include, but are not limited to, PEG-diacylglycerol (DAG), PEG-dialkyloxypropyl (DAA), PEG-phospholipid, PEG-ceramide (Cer), or mixtures thereof. It can be a polyethylene glycol (PEG)-lipid containing. PEG-DAA conjugates are, for example, PEG-dilauryloxypropyl (Ci2), PEG-dimyristyloxypropyl (Ci4), PEG-dipalmityloxypropyl (Ci6), or PEG-distearyloxypropyl (Ci). 8). The conjugated lipid that prevents particle aggregation can be from 0 mol% to about 20 mol%, or 2 mol% of the total lipids present in the particle.

In some embodiments, the nucleic acid-lipid particles further comprise cholesterol, such as from about 10 mol% to about 60 mol% or about 48 mol% of the total lipids present in the particle.

In one embodiment, the lipidoid ND98.4HCl (MW 1487) (U.S. Patent Application No. 12/056,230, filed March 26, 2008, incorporated herein by reference) ), cholesterol (Sigma-Aldrich), and PEG-Ceramide C16 (Avanti Polar Lipids) can be used to prepare lipid-dsRNA nanoparticles (ie, LNP01 particles). Stock solutions of each in ethanol can be prepared as follows: ND98, 133 mg/ml; cholesterol, 25 mg/ml, PEG-Ceramide C16, 100 mg/ml. ND98, cholesterol, and PEG-Ceramide C16 stock solutions can then be combined in a molar ratio of, for example, 42:48:10. The combined lipid solution is mixed with an aqueous dsRNA solution (e.g., in sodium acetate (pH 5)) such that the final ethanol concentration is about 35-45% and the final sodium acetate concentration is about 100-300 mM. obtain. Lipid-dsRNA nanoparticles usually form spontaneously upon mixing. Depending on the desired particle size distribution, the resulting nanoparticle mixture may be passed through a polycarbonate membrane (e.g., 100 nm cutoff) using a thermobarrel extruder, e.g., a Lipex Extruder (Northern Lipids, Inc). Can be pushed out. In some cases, the extrusion step can be omitted. Ethanol removal and simultaneous buffer exchange can be accomplished, for example, by dialysis or tangential flow filtration. The buffer may be, for example, phosphate buffered saline (PBS) at about pH 7, such as about pH 6.9, about pH 7.0, about pH 7.1, about pH 7.2, about pH 7.3, or about pH 7.4. ) can be exchanged with

LNP01 formulations are described, for example, in International Application Publication No. WO 2008/042973, which is incorporated herein by reference.

Additional exemplary lipid-dsRNA formulations are listed in Table 1.

DSPC: Distearoyl phosphatidylcholine DPPC: Dipalmitoylphosphatidylcholine PEG-DMG: PEG-didimyristoyl glycerol (C14-PEG, or PEG-C14) (PEG with an average molecular weight of 2000)
PEG-DSG: PEG-distyrylglycerol (C18-PEG, or PEG-C18) (PEG with an average molecular weight of 2000)
PEG-cDMA: PEG-carbamoyl-1,2-dimyristyloxypropylamine (PEG with average molecular weight of 2000)
A formulation containing SNALP (l,2-dilinolenyloxy-N,N-dimethylaminepropane (DLinDMA)) is described in International Publication No. 1, filed April 15, 2009, which is incorporated herein by reference. It is described in the 2009/127060 pamphlet.

Formulations containing XTC are described, for example, in WO 2010/088537, the entire contents of which are incorporated herein by reference.

Formulations containing MC3 are described, for example, in U.S. Patent Application Publication No. 2010/0324120, filed June 10, 2010, the entire contents of which are incorporated herein by reference. .

Formulations containing ALNY-100 are described, for example, in WO 2010/054406, the entire contents of which are incorporated herein by reference.

Formulations containing C12-200 are described in WO 2010/129709, the entire contents of which are incorporated herein by reference.

Compositions and formulations for oral administration include powders or granules, microparticles, nanoparticles, suspensions, or solutions in water or non-aqueous media, capsules, gel capsules, sachets, tablets or mini-tablets. . Thickeners, flavoring agents, diluents, emulsifiers, dispersing aids or binders may be desired. In some embodiments, an oral formulation is one in which a dsRNA featuring the invention is administered with one or more permeation enhancers, surfactants, and chelating agents. Suitable surfactants include fatty acids and/or esters or salts thereof, bile acids and/or salts thereof. Suitable bile acids/salts include chenodeoxycholic acid (CDCA) and ursodeoxychenodeoxycholic acid (UDCA), cholic acid, dehydrocholic acid, deoxycholic acid, glycolic acid, glycolic acid, glycodeoxycholic acid, taurocholic acid, taurodeoxy Mention may be made of cholic acid, sodium tauro-24,25-dihydro-fusidate and sodium glycodihydrofusidate. Suitable fatty acids include arachidonic acid, undecanoic acid, oleic acid, lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein, dilauric acid, Examples include glyceryl 1-monocaprate, 1-dodecyl azacycloheptan-2-one, acylcarnitine, acylcholine, or monoglycerides, diglycerides, or pharmaceutically acceptable salts thereof (eg, sodium). In some embodiments, combinations of penetration enhancers are used, such as fatty acids/salts in combination with bile acids/salts. One exemplary combination is the sodium salt of lauric acid, capric acid, and UDCA. Additional penetration enhancers include polyoxyethylene-9-lauryl ether, polyoxyethylene-20-cetyl ether. The DsRNA that characterizes the invention can be delivered orally in granular form, including spray-dried particles or complexed to form micro- or nanoparticles. dsRNA complexing agents include poly-amino acids; polyimines; polyacrylates; polyalkyl acrylates, polyoxetanes, polyalkyl cyanoacrylates; cationized gelatin, albumin, starch, acrylates, polyethylene glycol (PEG) and starch; polyalkyl cyanoacrylates ; DEAE-derivatized polyimines, pullulan, cellulose and starch. Suitable complexing agents include chitosan, N-trimethylchitosan, poly-L-lysine, polyhistidine, polyornithine, polyspermine, protamine, polyvinylpyridine, polythiodiethylaminomethylethylene P (TDAE), polyaminostyrene (e.g. p-amino), poly(methylcyanoacrylate), poly(ethylcyanoacrylate), poly(butylcyanoacrylate), poly(isobutylcyanoacrylate), poly(isohexylcyano(cynao)acrylate), DEAE-methacrylate, DEAE- Hexyl acrylate, DEAE-acrylamide, DEAE-albumin and DEAE-dextran, polymethyl acrylate, polyhexyl acrylate, poly(D,L-lactic acid), poly(DL-lactic-co-glycolic acid (PLGA)), alginate, and Oral formulations for dsRNA and their formulations are described in U.S. Patent No. 6,887,906, U.S. Patent Application Publication No. 20030027780, each of which is incorporated herein by reference. and US Pat. No. 6,747,014.

Compositions and preparations for parenteral, intraparenchymal (into the brain), intrathecal, intraventricular or intrahepatic administration may include sterile aqueous solutions, including buffered, diluted agents, and other suitable additives such as, but not limited to, penetration enhancers, carrier compounds, and other pharmaceutically acceptable carriers or excipients.

Pharmaceutical compositions of the invention include, but are not limited to, solutions, emulsions, and liposome-containing formulations. These compositions may be produced from a variety of components including, but not limited to, preformed liquids, self-emulsifying solids, and self-emulsifying semi-solids. In treating liver diseases such as hepatic carcinoma, liver-targeted formulations are particularly preferred.

Pharmaceutical formulations of the invention, which may conveniently be presented in unit dosage form, can be prepared according to conventional techniques well known in the pharmaceutical industry. Such techniques include the step of bringing into association the active ingredient with a pharmaceutical carrier or excipient. In general, the formulations are prepared by uniformly and intimately bringing the active ingredient into association with liquid carriers or finely divided solid carriers, or both, and then, if necessary, shaping the product.

The compositions of the invention may be formulated into any of a number of possible dosage forms, including, but not limited to, tablets, capsules, gel capsules, liquid syrups, soft gels, suppositories, and enemas. Compositions of the invention may also be formulated as suspensions in aqueous, non-aqueous or mixed media. Aqueous suspensions may further contain substances that increase the viscosity of the suspension, including, for example, sodium carboxymethyl cellulose, sorbitol and/or dextran. The suspension may also contain stabilizers.

C. Further formulations i. Emulsions Compositions of the invention can be prepared and formulated as emulsions. Emulsions are typically heterogeneous systems in which one liquid is dispersed in another liquid in the form of droplets usually greater than 0.1 μm in diameter (e.g., Ansel's Pharmaceutical Dosage Forms and Drugs). Delivery Systems, Allen, LV., Popovich NG., and Ansel HC., 2004, Lippincott Williams & Wilkins (8th ed.), New York, NY; Idson, in Pharma Ceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988 , Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199; Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 19 88, Marcel Dekker, Inc., New York, N.Y., Volume 1, p. 245; Block in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York , N.Y., volume 2, p.335 ; Higuchi et al., in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., 1985, p. 301). Emulsions are often bilayer systems containing two immiscible liquid phases intimately mixed and dispersed with each other. Generally, emulsions can be either of the water-in-oil (w/o) or oil-in-water (o/w) variety. When the aqueous phase is finely divided and dispersed in the bulk oil phase as microdroplets, the resulting composition is called a water-in-oil (w/o) emulsion. Alternatively, if the oil phase is finely divided and dispersed in the aqueous phase of the mass as microdroplets, the resulting composition is called an oil-in-water (o/w) emulsion. Emulsions can contain additional components in addition to the dispersed phase and the active drug, which components can be present as solutions in an aqueous phase, an oil phase, or as separate phases themselves. Pharmaceutical excipients such as emulsifiers, stabilizers, dyes, and antioxidants may also be present in the emulsion if desired. Pharmaceutical emulsions can be multiple emulsions consisting of three or more phases, such as in the case of oil-in-water-in-oil (o/w/o) and water-in-oil-in-water (w/o/w) emulsions. Such complex formulations often offer certain advantages that simple two-component emulsions do not. Multiemulsions in which individual oil droplets of an o/w emulsion enclose smaller water droplets constitute a w/o/w emulsion. Similarly, a system of oil droplets enclosed in globules of water stabilized in a continuous phase of oil provides an o/w/o emulsion.

Emulsions are characterized by having little or no thermodynamic stability. Often the dispersed or discontinuous phase of an emulsion is well dispersed in the external or continuous phase and maintained in this form by means of emulsifiers or through the viscosity of the formulation. Any of the emulsion phases can be semi-solid or solid, as in emulsion-type ointment bases and creams. Other means of stabilizing emulsions include the use of emulsifiers that can be incorporated into either phase of the emulsion. Emulsifiers can be broadly classified into four categories: synthetic surfactants, natural emulsifiers, absorption bases, and finely dispersed solids (e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich NG., and Ansel HC., 2004, Lippincott Williams & Wilkins (8th ed.), New York, NY; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieg. er and Banker (Eds.), 1988, Marcel Dekker, Inc., New York , N.Y., volume 1, p. 199).

Synthetic surfactants, also known as surfactants, have found wide applicability in the formulation of emulsions and are reviewed in the literature (e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems , Allen, LV., Popovich NG., and Ansel HC., 2004, Lippincott Williams & Wilkins (8th ed.), New York, NY; Rieger, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 285; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), Marcel Dekk er, Inc., New York, N.Y. , 1988, volume 1, p. 199). Surfactants are usually amphipathic and include a hydrophilic and a hydrophobic portion. The ratio of hydrophilicity to hydrophobicity of a surfactant is referred to as the hydrophilic/lipophilic balance (HLB) and is a valuable tool in the classification and selection of surfactants during formulation preparation. Surfactants can be classified into different types, namely nonionic, anionic, cationic and amphoteric, based on the nature of the hydrophilic group (see, for example, Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich NG., and Ansel HC., 2004, Lippincott Williams & Wilkins (8th ed.), New York, NY Rieger, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc. , New York, N.Y., volume 1, p. 285).

Naturally occurring emulsifiers used in emulsion formulations include lanolin, beeswax, phosphatides, lecithin and acacia. Absorbent bases, like anhydrous lanolin and hydrophilic petrolatum, possess hydrophilic properties that take up water to form a w/o emulsion but still maintain their semi-solid consistency. Micronized solids are used as good emulsifiers, especially in surfactant combinations and in viscous formulations. These include polar inorganic solids such as heavy metal hydroxides, non-swelling clays such as bentonite, attapulgite, hectorite, kaolin, montmorillonite, colloidal aluminum silicate and colloidal magnesium aluminum silicate, pigments, and non-swelling clays such as carbon. Mention may be made of polar solids or glyceryl tristearate.

A wide variety of non-emulsifying materials are also included in emulsion formulations and contribute to the properties of the emulsion. These include fats, oils, waxes, fatty acids, fatty alcohols, fatty esters, wetting agents, hydrophilic colloids, preservatives and antioxidants (Block, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds. ), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 335; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds. ), 1988, Marcel Dekker, Inc. New York, N.Y., volume 1, p. 199).

Hydrophilic colloids or hydrocolloids include naturally occurring gums and synthetic polymers such as polysaccharides (e.g. acacia, agar, alginic acid, carrageenan, guar gum, karaya gum, and tragacanth), cellulose derivatives (e.g. carboxymethyl cellulose and carboxypropyl cellulose). ), and synthetic polymers such as carbomers, cellulose ethers, and carboxyvinyl polymers. These are colloidal solutions that stabilize emulsions by dispersing or swelling in water to form a strong interfacial film around the dispersed phase droplets and by increasing the viscosity of the external phase. form.

These formulations often incorporate preservatives because emulsions often contain a number of ingredients such as carbohydrates, proteins, sterols and phosphatides that can readily support microbial growth. Commonly used preservatives included in formulations include methylparaben, propylparaben, quaternary ammonium salts, benzalkonium chloride, esters of p-hydroxybenzoic acid, and boric acid. Antioxidants are also commonly added to emulsion formulations to prevent spoilage of the formulation. Antioxidants used are free radical scavengers such as tocopherols, alkyl gallates, butylated hydroxyanisole, butylated hydroxytoluene, or reducing agents such as ascorbic acid and sodium metabisulfite, as well as citric acid, tartaric acid and lecithin. can be an antioxidant synergist.

The application of emulsion formulations via the dermal, oral and parenteral routes and methods of their manufacture are reviewed in the literature (e.g. Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich NG., and Ansel HC., 2004, Lippincott Williams & Wilkins (8th ed.), New York, NY; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger a. nd Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.D. Y., volume 1, p. 199). Emulsion formulations for oral delivery are very widely used due to their ease of formulation and effectiveness in terms of absorption and bioavailability (e.g., Ansel's Pharmaceutical Dosage Forms and Drugs). Delivery Systems, Allen, LV., Popovich NG., and Ansel HC., 2004, Lippincott Williams & Wilkins (8th ed.), New York, NY; Rosoff, in Pharm. Aceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988 , Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 198 8, Marcel Dekker, Inc., New York, (See NY, volume 1, p. 199). Mineral oil-based laxatives, oil-soluble vitamins, and high-fat nutritional formulations are among the materials commonly administered orally as o/w emulsions.

ii. Microemulsions In one embodiment of the invention, the iRNA and nucleic acid compositions are formulated as microemulsions. A microemulsion may be defined as a system of water, oil, and amphiphile that is a single optically isotropic and thermodynamically stable liquid solution (e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich NG., and Ansel HC., 2004, Lippincott Williams & Wilkins (8th ed.), New York, NY; Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245). Typically, microemulsions are made by first dispersing the oil in an aqueous surfactant solution and then adding a sufficient amount of a fourth component, generally an alcohol of intermediate chain length, to form a clear system. Prepared by Microemulsions are therefore described as thermodynamically stable, isotropically transparent dispersions of two immiscible liquids stabilized by an interfacial film of surface-active molecules (Leung and Shah , in: Controlled Release of Drugs: Polymers and Aggregate Systems, Rosoff, M., Ed., 1989, VCH Publishers, New York, pp. 185-215). Microemulsions are typically prepared using a combination of three to five components including oil, water, surfactants, cosurfactants, and electrolytes. Whether a microemulsion is of the water-in-oil (w/o) or oil-in-water (o/w) type depends on the properties of the oil and surfactant used and the polar head of the surfactant molecule. and the structure and geometric packing of the hydrocarbon tail (Schott, in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., 1985, p. 271 ).

Phenomenological approaches using phase diagrams have been extensively studied and have provided those skilled in the art with extensive knowledge of how to formulate microemulsions (e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen , LV., Popovich NG., and Ansel HC., 2004, Lippincott Williams & Wilkins (8th ed.), New York, NY; Rosoff, in Pharmaceutical Dosage Forms , Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245; Block, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker , Inc., New York, N.Y. , volume 1, p. 335). Compared to conventional emulsions, microemulsions offer the advantage of solubilizing water-insoluble drugs in the formulation of thermodynamically stable droplets that form spontaneously.

Surfactants used in the preparation of microemulsions include, but are not limited to, ionic surfactants, nonionic surfactants, Brij 96, polyoxyethylene, alone or in combination with cosurfactants. Oleyl ether, polyglycerol fatty acid ester, tetraglycerol monolaurate (ML310), tetraglycerol monooleate (MO310), hexaglycerol monooleate (PO310), hexaglycerol pentaoleate (PO500), decaglycerol monocaprate (MCA750) ), decaglycerol monooleate (MO750), decaglycerol sequioleate (SO750), and decaglycerol decaoleate (DAO750). Co-surfactants, which are usually short-chain alcohols such as ethanol, 1-propanol, and 1-butanol, penetrate into the surfactant film and are thus impregnated by the void spaces created between the surfactant molecules. By forming an ordered film, it plays a role in increasing interfacial fluidity. However, microemulsions can be prepared without the use of co-surfactants, and alcohol-free self-emulsifying microemulsion systems are known in the art. The aqueous phase can typically be, but is not limited to, water, an aqueous solution of a drug, glycerol, PEG300, PEG400, polyglycerol, propylene glycol, and ethylene glycol derivatives. The oil phase may include, but is not limited to, Captex 300, Captex 355, Capmul MCM, fatty acid esters, medium chain (C8-C12) mono-, di-, and tri-glycerides, polyoxyethylated glyceryl fatty acid esters, fatty alcohols, polyglycolated Materials such as glycerides, saturated polyglycolated C8-C10 glycerides, vegetable oils and silicone oils can be included.

Microemulsions are of particular interest from the standpoint of drug solubilization and enhanced drug absorption. Lipid-based microemulsions (both o/w and w/o) have been proposed to improve the oral bioavailability of drugs containing peptides (e.g., U.S. Pat. No. 6,191,105; U.S. Pat. No. 7,063,860, U.S. Patent No. 7,070,802, U.S. Patent No. 7,157,099, Constantinides et al., Pharmaceutical Research, 1994, 11, 1385-1390; Ritschel , Meth. Find. Exp. Clin. Pharmacol., 1993, 13, 205). Microemulsions offer improved drug solubilization, protection of drugs from enzymatic hydrolysis, possible enhancement of drug absorption due to surfactant-induced alterations in membrane fluidity and permeability, ease of preparation, and solid dosage forms. (e.g., U.S. Pat. No. 6,191,105; U.S. Pat. No. 7,063,860; U.S. Patent No. 7,070,802, U.S. Patent No. 7,157,099, Constantinides et al., Pharmaceutical Research, 1994, 11, 1385; Ho et al., J. Pharm. Sci., 1996, 85, 138-143). In many cases, microemulsions can form spontaneously when the components of the microemulsion are brought together at ambient temperature. This can be particularly advantageous when formulating heat-labile drugs, peptides or iRNAs. Microemulsions are also useful for cost-effective delivery of active ingredients in both cosmetic and pharmaceutical applications. It is expected that the microemulsion compositions and formulations of the present invention will promote increased systemic absorption of iRNA and nucleic acids from the gastrointestinal tract and improved local cellular uptake of iRNA and nucleic acids.

The microemulsions of the present invention also include additional components and additives such as sorbitan monostearate (Grill 3), Labrasol, and permeation enhancers to improve the properties of the formulation and to improve the properties of the present invention. Absorption of iRNA and nucleic acids can be improved. The penetration enhancers used in the microemulsions of the present invention can be classified as belonging to one of five broad categories--surfactants, fatty acids, bile salts, chelating agents, and non-chelating non-chelating agents. Surfactants (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92). Each of these classes is discussed above.

iii. Microparticles The iRNA agents of the invention may be incorporated into particles, such as microparticles. Microparticles may be produced by spray drying, but may also be produced by other methods including freeze drying, evaporation, fluid bed drying, vacuum drying, or a combination of these techniques.

iv. Penetration Enhancers In one embodiment, the present invention employs various penetration enhancers to effect efficient delivery of nucleic acids, particularly iRNA, to the skin of animals. Most drugs exist in solution in both ionized and non-ionized forms. However, usually only lipophilic or lipophilic drugs readily cross cell membranes. It has been discovered that even non-lipophilic drugs can cross cell membranes if the membrane being crossed is treated with a penetration enhancer. In addition to assisting in the diffusion of non-lipophilic drugs across cell membranes, penetration enhancers also improve the permeability of lipophilic drugs.

Penetration enhancers can be classified as belonging to one of five broad categories: surfactants, fatty acids, bile salts, chelating agents, and non-chelating non-surfactants (see, eg, Malmsten, M. Surfactants and polymers in drug delivery, Informa Health Care, New York, NY, 2002; Lee et al., Critical Reviews in Therapeutic D rug Carrier Systems, 1991, p. 92). Each of the above types of penetration enhancers are described in more detail below.

Surfactants (or "surfactants"), when dissolved in an aqueous solution, reduce the surface tension of the solution or the interfacial tension between the aqueous solution and another liquid, improving the absorption of iRNA across mucosal membranes. It is a chemical substance that causes this result. In addition to bile salts and fatty acids, these penetration enhancers include, for example, sodium lauryl sulfate, polyoxyethylene-9-lauryl ether and polyoxyethylene-20-cetyl ether (see, for example, Malmsten, M. Surfactants and polymers). in drug delivery, Informa Health Care, New York, NY, 2002; Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92); and perfluorinated compound emulsions such as FC-43 (see Takahashi et al. ., J. Pharm. Pharmacol., 1988, 40, 252).

Various fatty acids and their derivatives that act as penetration enhancers include, for example, oleic acid, lauric acid, capric acid (n-decanoic acid), myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate. , tricaprate, monoolein (1-monooleyl-rac-glycerol), dilaurin, caprylic acid, arachidonic acid, glycerol 1-monocaprate, 1-dodecyl azacycloheptan-2-one, acylcarnitine, acylcholine, their C1~ 20 alkyl esters (e.g., methyl, isopropyl, and t-butyl), and their mono- and diglycerides (i.e., oleate, laurate, caprate, myristate, palmitate, stearate, linoleate, etc.) (e.g., Touitou, E. ., et al. Enhancement in Drug Delivery, CRC Press, Danvers, MA, 2006; Lee et al., Critical Reviews in Therapeutic Drug Carrier Syst ems, 1991, p. 92; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33; see El Hariri et al., J. Pharm. Pharmacol., 1992, 44, 651-654).

Physiological roles of bile include promoting the dispersion and absorption of lipids and fat-soluble vitamins (e.g., Malmsten, M. Surfactants and polymers in drug delivery, Informa Health Care, New York, NY, 2002; Brunton ,Chapter 38 in: Goodman & Gilman's The Pharmacological Basis of Therapeutics, 9th Ed., Hardman et al. Eds., McGraw-Hill, New York, 1996, pp.93 4-935). Various natural bile salts and their synthetic derivatives act as penetration enhancers. Accordingly, the term "bile salts" includes any of the natural components of bile as well as any of their synthetic derivatives. Suitable bile salts include, for example, cholic acid (or its pharmaceutically acceptable sodium salt, sodium cholate), dehydrocholic acid (sodium dehydrocholic acid), deoxycholic acid (sodium deoxycholate), glycolic acid. (glucholic acid) (sodium glucholate), glycolic acid (sodium glycocholate), glycodeoxycholic acid (sodium glycodeoxycholate), taurocholic acid (sodium taurocholate), taurodeoxycholic acid (sodium glycolate) sodium cholate), chenodeoxycholic acid (sodium chenodeoxycholate), ursodeoxycholic acid (UDCA), sodium tauro-24,25-dihydro-fucidate (STDHF), sodium glycodihydrofusidate and polyoxyethylene-9-lauryl ether ( POE) (e.g., Malmsten, M. Surfactants and polymers in drug delivery, Informa Health Care, New York, NY, 2002; Lee et al., Critical Rev. ies in Therapeutic Drug Carrier Systems, 1991, page 92; Swinyard, Chapter 39 In: Remington's Pharmaceutical Sciences, 18th Ed., Gennaro, ed., Mack Publishing Co., Easton, Pa., 1990, pp. 782-783; Muran ishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7 , 1-33; Yamamoto et al., J. Pharm. Exp. Ther., 1992, 263, 25; Yamashita et al., J. Pharm. Sci., 1990, 79, 579-583).

A chelating agent used in connection with the present invention may be defined as a compound that removes metal ions from solution by forming a complex with the metal ions, resulting in improved absorption of iRNA across mucous membranes. . Regarding the use of chelating agents as penetration enhancers in the present invention, since most characterized DNA nucleases require divalent metal ions for catalysis and are therefore inhibited by chelating agents, chelating agents can be It has the added advantage of also acting as a DNase inhibitor (Jarrett, J. Chromatogr., 1993, 618, 315-339). Suitable chelating agents include, but are not limited to, disodium ethylenediaminetetraacetate (EDTA), citric acid, salicylates (e.g., sodium salicylate, 5-methoxysalicylate and homovanilate), N-acyl derivatives of collagen. , laureth-9 and N-aminoacyl derivatives (enamines) of β-diketones (for example, Katdare, A. et al., Experimental development for pharmaceutical, biotechnology, and drug delivery, CRC). Press, Danvers, MA, 2006; Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p.92; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p.92; Burier Systems, 1990, 7, 1-33; Burur et al., J. Control Rel., 1990, 14, 43-51).

As used herein, a non-chelating, non-surfactant penetration-enhancing compound exhibits little activity as a chelating agent or surfactant, but nevertheless enhances the absorption of iRNA across the gastrointestinal mucosa. (See, eg, Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33). Penetration enhancers of this type include, for example, unsaturated cyclic ureas, 1-alkyl- and 1-alkenylazacyclo-alkanone derivatives (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92); and non-steroidal anti-inflammatory drugs such as diclofenac sodium, indomethacin and phenylbutazone (Yamashita et al., J. Pharm. Pharmacol., 1987, 39, 621-626).

Agents that promote iRNA uptake at the cellular level may also be added to the pharmaceutical compositions and other compositions of the invention. For example, cationic lipids such as lipofectin (US Pat. No. 5,705,188 to Junichi et al.), cationic glycerol derivatives, and polycationic molecules such as polylysine (International Publication of PCT Applications of Lollo et al. No. 97/30731 pamphlet) is also known to promote cellular uptake of dsRNA. Examples of commercially available transfection reagents include, inter alia, Lipofectamine™ (Invitrogen; Carlsbad, CA), Lipofectamine 2000™ (Invitrogen; Carlsbad, CA), 293fectin™ (Invitrogen; gen; Carlsbad, CA) , Cellfectin™ (Invitrogen; Carlsbad, CA), DMRIE-C™ (Invitrogen; Carlsbad, CA), FreeStyle™ MAX (Invitrogen; Carlsbad, CA), Lipofecta mine(TM) 2000 CD (Invitrogen; Carlsbad , CA), Lipofectamine™ (Invitrogen; Carlsbad, CA), iRNAMAX (Invitrogen; Carlsbad, CA), Oligofectamine™ (Invitrogen; Carlsbad, CA), Opt ifect™ (Invitrogen; Carlsbad, CA), X -tremeGENE Q2 Transfection Reagent (Roche; Grenzacherstrasse, Switzerland), DOTAP Liposomal Transfection Reagent (Grenzach DOSPER Liposomal Transfection Reagent (Grenzacherstrasse, Switzerland), or Fugene (Grenzacherstrasse, Switzerland) , Switzerland), Transfectam® Reagent (Promega; Madison, WI), TransFast™ Transfection Reagent (Promega; Madison, WI), Tfx™-20 Reagent (Promega; Madison, WI), Tfx™-50 Reagent (Pr omega; Madison, WI), DreamFect (trademark) (OZ Biosciences; Marseille, France), EcoTransfect (OZ Biosciences; Marseille, France), TransPass ^a D1 Transfection Reagent (N ew England Biolabs; Ipswich, MA, USA), LyoVec(TM)/LipoGen(TM)(Invitrogen ; San Diego, CA, USA), Perfectin Transfection Reagent (Genlantis; San Diego, CA, USA), NeuroPORTER Transfection Reagent (Genlantis; n Diego, CA, USA), GenePORTER Transfection reagent (Genlantis; San Diego, CA, USA) , GenePORTER 2 Transfection reagent (Genlantis; San Diego, CA, USA), Cytofectin Transfection Reagent (Genlantis; San Diego, CA, USA), BaculoPORTER Transfection Reagent (Genlantis; San Diego, CA, USA), TroganPORTER™transfection Reagent (Genlantis; San Diego, CA, USA), RiboFect (Bioline; Taunton, MA, USA), PlasFect (Bioline; Taunton, MA, USA), UniFECTOR (B-Bridge International nal; Mountain View, CA, USA), SureFECTOR ( B-Bridge International; Mountain View, CA, USA), or HiFect™ (B-Bridge International, Mountain View, CA, USA).

Other agents can be used to enhance the penetration of the administered nucleic acids, including glycols such as ethylene glycol and propylene glycol, pyrroles such as 2-pyrrole, azones, and terpenes such as limonene and menthone.

v. Carriers Certain compositions of the invention also incorporate carrier compounds in the formulation. As used herein, a "carrier compound" or "carrier" can be inert (i.e., not possessing biological activity), but can, for example, degrade biologically active nucleic acids or Can refer to a nucleic acid or an analog thereof that is recognized as a nucleic acid by an in vivo process that reduces the bioavailability of a biologically active nucleic acid by promoting its removal from the circulation. Co-administration of a nucleic acid and a carrier compound, typically with an excess of the latter substance, may result in an increase in liver, kidney or other extracirculatory reservoirs (possibly due to competition between the carrier compound and the nucleic acid for common receptors). The amount of nucleic acid recovered in the extracirculatory reservoir may be substantially reduced. For example, partial phosphorothioate recovery within liver tissue is possible if it is co-administered with polyinosinic acid, dextran sulfate, polycytidic acid or 4-acetamido-4'isothiocyano-stilbene-2,2'-disulfonic acid. (Miyao et al., DsRNA Res. Dev., 1995, 5, 115-121; Takakura et al., DsRNA & Nucl. Acid Drug Dev., 1996, 6, 177-183.

vi. Excipients In contrast to carrier compounds, "pharmaceutical carriers" or "excipients" refer to pharmaceutically acceptable solvents, suspending agents, or any Other pharmacologically inert vehicles. Excipients can be liquids or solids and have the desired bulk, consistency, etc. when combined with the nucleic acid and other prescribed components of the pharmaceutical composition, taking into account the intended method of administration. selected to provide. Typical pharmaceutical carriers include, but are not limited to, binders such as pregelatinized corn starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose; fillers such as lactose and other sugars, microcrystalline cellulose, pectin, gelatin, etc. , calcium sulfate, ethyl cellulose, polyacrylate or calcium hydrogen phosphate); lubricants (such as magnesium stearate, talc, silica, colloidal silicon dioxide, stearic acid, metal stearates, hydrogenated vegetable oils, corn starch, Tablet disintegrants (eg, starch, sodium starch glycolate, etc.); and wetting agents (eg, sodium lauryl sulfate, etc.).

Suitable organic or inorganic excipients that do not deleteriously react with the nucleic acids and are pharmaceutically acceptable for parenteral administration may also be used in formulating the compositions of the invention. Suitable pharmaceutically acceptable carriers include, but are not limited to, water, saline, alcohol, polyethylene glycol, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone. Examples include.

Formulations for topical administration of nucleic acids can include sterile and non-sterile aqueous solutions, non-aqueous solutions, or solutions of the nucleic acids in liquid or solid oil bases in common solvents such as alcohols. Solutions may also include buffers, diluents and other suitable additives. Any suitable organic or inorganic excipient that does not deleteriously react with the nucleic acid and is pharmaceutically acceptable for parenteral administration may be used.

Suitable pharmaceutically acceptable excipients include, but are not limited to, water, saline, alcohol, polyethylene glycol, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethyl cellulose, Examples include polyvinylpyrrolidone.

vii. Other Components The compositions of the present invention may also contain other adjunct components conventionally found in pharmaceutical compositions, at their art-established usage levels. Thus, for example, the composition may contain further compatible pharmaceutically active materials, such as, for example, antipruritics, astringents, local anesthetics or anti-inflammatory agents, or dyes, flavoring agents, Additional materials useful in physically formulating the various dosage forms of the compositions of the present invention may be included, such as preservatives, antioxidants, opacifiers, thickeners, and stabilizers. However, when added, these materials need not unduly interfere with the biological activity of the components of the compositions of the invention. The formulation may be sterilized and, if desired, may contain adjuvants that do not deleteriously interact with the nucleic acids of the formulation, such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts that affect osmotic pressure, and buffers. It is mixed with liquid, colorants, seasonings and/or aromatic substances.

Aqueous suspensions may contain substances that increase the viscosity of the suspension, including, for example, sodium carboxymethyl cellulose, sorbitol and/or dextran. The suspension may also contain stabilizers.

In some embodiments, the pharmaceutical compositions featured in this invention include (a) one or more iRNA compounds; and (b) one or more agents that function by a non-iRNA mechanism and are useful for treating HBV infection. including. Examples of such agents include, but are not limited to, antiviral agents aimed at suppressing or destroying HBV by interfering with viral replication; and antiviral agents aimed at helping the human immune system mount defenses against the virus. Examples include immunomodulatory drugs. In contrast, immunomodulatory drugs such as corticosteroids, or adenine arabinoside, acyclovir, or dideoxyinosine, which induce enhanced expression of viruses and viral antigens and suppression of T lymphocyte function, are effective in treating chronic hepatitis B. Not beneficial for treatment. Suitable agents are discussed in more detail below.

The toxicity and therapeutic efficacy of such compounds can be determined, for example, in cell cultures or in experiments to determine the LD50 (dose lethal to 50% of a population) and ED50 (dose therapeutically effective to 50% of a population). It can be determined by standard pharmaceutical procedures in animals. The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50/ED50. Compounds exhibiting high therapeutic indices are preferred.

The data obtained from cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of the compositions featured herein generally lies within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending on the dosage form used and the route of administration used. For any compound used in the methods featured in this invention, the therapeutically effective dose can be estimated initially from cell culture assays. The dose is determined in cell culture to include the IC50 (i.e., the concentration of the test compound that achieves half-maximal inhibition of symptoms) of the compound, or, if appropriate, the polypeptide product of the target sequence, in the circulating plasma. A range of concentrations can be formulated to be achieved in an animal model (eg, to achieve a reduced concentration of the polypeptide). Such information can be used to more accurately determine useful doses in humans. Levels in plasma can be measured, for example, by high performance liquid chromatography.

In addition to their administration as described above, the iRNAs that feature the invention may be administered in combination with other known agents effective in the treatment of pathological processes mediated by HBV expression. In either case, the administering physician will determine the amount and administration of iRNA based on the observed results and using standard efficacy measures known in the art or described herein. You can adjust the time.

VII. METHODS OF THE INVENTION The present invention provides methods for treating subjects who have or are susceptible to developing HBV infection and/or HBV-related diseases, disorders, and/or conditions (e.g., CHB). Treatment and prevention methods are provided that include the step of administering a composition comprising an iRNA agent of the invention, a pharmaceutical composition comprising an iRNA agent, or a vector comprising an iRNA.

The methods of the invention are useful in treating subjects with HBV infection, eg, subjects who could benefit from reducing HBV gene expression and/or HBV replication. In one aspect, the invention provides a method of reducing the level of Hepatis B virus ccc DNA in a subject infected with HBV. In another aspect, the invention provides a method of reducing levels of HBV antigens, such as HBsAg and/or HBeAg, in a subject infected with HBV. In another aspect, the invention provides a method of reducing HBV viral load in a subject infected with HBV. The present invention also provides methods for reducing levels of alanine aminotransferase (ALT) and/or aspartate aminotransferase (AST) in a subject infected with HBV. In one aspect, the invention provides a method of increasing the level of anti-HBV antibodies in a subject infected with HBV. In another aspect, the invention provides a method of treating a subject with an HBV infection. In one aspect, the present invention relates to HBV-related diseases, such as hepatitis D virus infection, delta hepatitis, acute hepatitis B; acute fulminant hepatitis B; chronic hepatitis B; liver fibrosis; end-stage liver disease; A method of treating a subject with cancer is provided. Furthermore, because HDV infection relies on essential helper functions provided by HBV for transmission, and subjects with HBV infection may also have HDV infection, the treatment methods described herein also reduce the risk of HDV infection and/or Also useful for treating subjects with HDV-related disorders, such as hepatitis B virus infection, chronic hepatitis B infection (CHB), chronic hepatitis B infection (CHB), liver cirrhosis, liver failure, and hepatocellular carcinoma (HCC). It is. The therapeutic methods (and uses) of the present invention include an iRNA agent of the present invention that targets an HBV gene, or a pharmaceutical composition comprising an iRNA agent of the present invention that targets an HBV gene, or an iRNA agent that targets an HBV gene. administering to a subject, eg, a human, a therapeutically effective amount of a vector of the invention comprising a vector of the invention.

In one aspect, the invention relates to at least one symptom in a subject having an HBV infection, such as the presence of serum and/or liver HBV ccc DNA, the presence of serum HBV DNA, the presence of serum and/or liver HBV antigens, such as HBsAg and/or or presence of HBeAg, elevated ALT, elevated AST, absence or low levels of anti-HBV antibodies, liver injury; cirrhosis; hepatitis D virus infection, delta hepatitis, acute hepatitis B; acute fulminant hepatitis B; Chronic hepatitis; liver fibrosis; end-stage liver disease; hepatocellular carcinoma; serum sickness-like syndrome; anorexia; nausea; vomiting; mild fever; muscle pain; fatigue; impaired taste and smell (food and tobacco aversion); and/or right upper quadrant and epigastric pain (intermittent, mild to moderate); hepatic encephalopathy; somnolence; disturbed sleep patterns; mental confusion; coma; ascites; gastrointestinal bleeding; coagulopathy; jaundice; hepatomegaly ( Splenomegaly; palmar erythema; spider nevus; muscle wasting; spider hemangioma; vasculitis; variceal hemorrhage; peripheral edema; gynecomastia; testicular atrophy; abdominal collateral veins (caput medusa); high levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST), in the range of 1000-2000 IU/mL, but also 100 times higher than the upper limit of normal range (ULN). ALT levels higher than AST levels; elevated γ-glutamyl transpeptidase (GGT) and alkaline phosphatase (ALP) levels (e.g., less than 3 times ULN); moderately low albumin levels; elevated serum iron levels; leukopenia (i.e., granulocytopenia); lymphocytosis; increased erythrocyte sedimentation rate (ESR); shortened red blood cell lifespan; hemolysis; thrombocytopenia; prolonged international normalized ratio (INR); serum and/or Presence of liver HBsAg, HBeAg, hepatitis B core antibody (anti-HBc) immunoglobulin M (IgM); hepatitis B surface antibody (anti-HBs), hepatitis B e antibody (anti-HBe), and/or HBV DNA; Elevated transferases (≤5 times ULN); ALT levels higher than AST levels; increased bilirubin levels, prolongation of prothrombin time (PT); hyperglobulinemia; anti-smooth muscle antibodies (ASMA) or anti-nuclear antibodies (ANA) presence of tissue non-specific antibodies such as (10-20%); presence of tissue-specific antibodies such as antibodies to the thyroid (10-20%); elevated levels of rheumatoid factor (RF); hyperbilirubinemia, PT prolongation, decreased platelet and white blood cell counts, AST levels higher than ALT levels; increased alkaline phosphatase (ALP) and GGT levels; lobular, with and associated inflammation with degenerative and reparative hepatocellular changes; mainly centrilobular Provides a method for preventing sexual necrosis. The method comprises administering a therapeutically effective amount of an iRNA agent of the invention, e.g., dsRNA, pharmaceutical composition, or vector, to a subject, such as a subject having an HBV infection or a subject having both an HBV and an HDV infection. Preventing at least one condition in a subject having a disorder that could benefit from reducing HBV gene expression.

In another aspect, the invention provides methods for treating a subject, such as a subject with an HBV infection or a subject with both an HBV and an HDV infection, who may benefit from reducing and/or inhibiting HBV gene expression. provides the use of a therapeutically effective amount of an iRNA agent of the invention.

In a further aspect, the invention provides that a subject, such as a subject with an HBV infection or a subject with both an HBV and an HDV infection, may benefit from the reduction and/or inhibition of HBV gene expression and/or HBV replication. iRNA agents of the invention that target HBV genes, such as dsRNAs that target HBV genes, in the manufacture of medicaments for the treatment of subjects and subjects with disorders, such as HBV-related diseases, that may benefit from reduction of HBV gene expression. Provided are uses of pharmaceutical compositions comprising iRNA agents for analysing.

In another aspect, the present invention provides methods for preventing at least one symptom in a subject suffering from a disorder that could benefit from reducing and/or inhibiting HBV gene expression and/or HBV replication. Provides for the use of iRNA, such as dsRNA.

In a further aspect, the invention provides a medicament for the prophylaxis of at least one condition in a subject suffering from a disorder that may benefit from the reduction and/or inhibition of HBV gene expression and/or HBV replication, such as an HBV-related disease. Provided is the use of an iRNA agent of the present invention in the production of.

In one embodiment, an iRNA agent that targets HBV is administered to a subject with an HBV infection or both HBV and HDV infection, and/or an HBV-related disease, when the dsRNA agent is administered to the subject, e.g. , expression of one or more HBV genes in blood or other tissues or body fluids, HBV ccc DNA levels, HBV antigen levels, HBV viral load levels, ALT, and/or AST of at least about 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29% , 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46 %, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 62%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79% , 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96 %, 97%, 98%, or at least about 99% or more.

In one embodiment, an iRNA agent that targets HBV is administered to a subject with an HBV infection or both HBV and HDV infection, and/or an HBV-related disease, when the dsRNA agent is administered to the subject, e.g. , the level of anti-HBV antibodies in the blood or other tissue or body fluid is at least about 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37% , 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54 %, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 62%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87% , 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least about 99% or more.

The methods and uses of the present invention may include approximately , 64, 68, 72, 76, or about 80 hours. In one embodiment, expression of the target HBV gene is maintained for an extended period of time, such as at least about 2, 3, 4, 5, 6, 7 or more days, such as about 1 week, 2 weeks, 3 weeks, or about 4 days. Declines over a week or more.

Administration of dsRNA according to the methods and uses of the invention can reduce the severity, signs, symptoms, and/or markers of HBV infection or both HBV and HDV infection and/or HBV-related diseases in patients with such diseases or disorders. may result in a reduction. "Reduction" in this context means a statistically significant reduction in such level. The reduction may be, for example, at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%. %, 80%, 85%, 90%, 95%, or about 100%.

The effectiveness of treatment or prevention of a disease is determined by, for example, disease progression, disease remission, symptom severity, pain reduction, quality of life, the dose of drug required to sustain treatment effects, and the level of disease markers. or any other measurable parameter appropriate to the given disease being treated or prevented. It is well within the ability of those skilled in the art to monitor the effectiveness of treatment or prophylaxis by measuring any one such parameter, or any combination of parameters. For example, the effectiveness of treating dyslipidemia can be evaluated by regular monitoring of, eg, LDL cholesterol, HDL cholesterol, and triglyceride levels. In a further example, the effectiveness of treating disorders of glucose control can be evaluated, for example, by regular monitoring of insulin and glucose levels. In another example, the effectiveness of obesity treatment can be evaluated, for example, by periodic monitoring of body mass index. In yet another example, the effectiveness of treating hypertension can be assessed, for example, by periodic monitoring of blood pressure. Comparison of early and later readings gives the physician an indication of whether the treatment is effective. It is well within the ability of those skilled in the art to monitor the effectiveness of treatment or prophylaxis by measuring any one such parameter, or any combination of parameters. In the context of administration of an iRNA or a pharmaceutical composition thereof that targets HBV, "effective against" a disease associated with HBV means that administration in a clinically appropriate manner improves symptoms, cures the disease, or cures the disease. at least statistically significant, such as mitigation, prolongation of life, improvement of quality of life, or other effects generally recognized as favorable by physicians skilled in the treatment of HBV infection and/or HBV-related diseases and associated causes. demonstrated beneficial effects in a significant proportion of patients.

Effectiveness of treatment or prevention is evidenced by a statistically significant improvement in one or more parameters of the disease state, or by worsening or failure of the originally expected symptoms to occur. By way of example, a favorable change of at least 10%, preferably at least 20%, 30%, 40%, 50% or more in a measurable parameter of the disease may indicate effective treatment. The efficacy of a given iRNA agent or formulation of that agent can also be determined using experimental animal models for a given disease, as is known in the art. When using experimental animal models, efficacy of treatment is demonstrated when a statistically significant reduction in markers or symptoms is observed.

For the target, about 0.01mg/kg, 0.02mg/kg, 0.03mg/kg, 0.04mg/kg, 0.05mg/kg, 0.1mg/kg, 0.15mg/kg, 0.2mg /kg, 0.25mg/kg, 0.3mg/kg, 0.35mg/kg, 0.4mg/kg, 0.45mg/kg, 0.5mg/kg, 0.55mg/kg, 0.6mg/kg , 0.65mg/kg, 0.7mg/kg, 0.75mg/kg, 0.8mg/kg, 0.85mg/kg, 0.9mg/kg, 0.95mg/kg, 1.0mg/kg, 1 .1mg/kg, 1.2mg/kg, 1.3mg/kg, 1.4mg/kg, 1.5mg/kg, 1.6mg/kg, 1.7mg/kg, 1.8mg/kg, 1.9mg /kg, 2.0mg/kg, 2.1mg/kg, 2.2mg/kg, 2.3mg/kg, 2.4mg/kg, 2.5mg/kg dsRNA, 2.6mg/kg dsRNA, 2.7mg /kg dsRNA, 2.8mg/kg dsRNA, 2.9mg/kg dsRNA, 3.0mg/kg dsRNA, 3.1mg/kg dsRNA, 3.2mg/kg dsRNA, 3.3mg/kg dsRNA, 3.4mg/kg kg dsRNA, 3.5mg/kg dsRNA, 3.6mg/kg dsRNA, 3.7mg/kg dsRNA, 3.8mg/kg dsRNA, 3.9mg/kg dsRNA, 4.0mg/kg dsRNA, 4.1mg/kg dsRNA, 4.2mg/kg dsRNA, 4.3mg/kg dsRNA, 4.4mg/kg dsRNA, 4.5mg/kg dsRNA, 4.6mg/kg dsRNA, 4.7mg/kg dsRNA, 4.8mg/kg dsRNA , 4.9mg/kg dsRNA, 5.0mg/kg dsRNA, 5.1mg/kg dsRNA, 5.2mg/kg dsRNA, 5.3mg/kg dsRNA, 5.4mg/kg dsRNA, 5.5mg/kg dsRNA, 5.6mg/kg dsRNA, 5.7mg/kg dsRNA, 5.8mg/kg dsRNA, 5.9mg/kg dsRNA, 6.0mg/kg dsRNA, 6.1mg/kg dsRNA, 6.2mg/kg dsRNA, 6 .3mg/kg dsRNA, 6.4mg/kg dsRNA, 6.5mg/kg dsRNA, 6.6mg/kg dsRNA, 6.7mg/kg dsRNA, 6.8mg/kg dsRNA, 6.9mg/kg dsRNA, 7. 0mg/kg dsRNA, 7.1mg/kg dsRNA, 7.2mg/kg dsRNA, 7.3mg/kg dsRNA, 7.4mg/kg dsRNA, 7.5mg/kg dsRNA, 7.6mg/kg dsRNA, 7.7mg /kg dsRNA, 7.8mg/kg dsRNA, 7.9mg/kg dsRNA, 8.0mg/kg dsRNA, 8.1mg/kg dsRNA, 8.2mg/kg dsRNA, 8.3mg/kg dsRNA, 8.4mg/kg kg dsRNA, 8.5mg/kg dsRNA, 8.6mg/kg dsRNA, 8.7mg/kg dsRNA, 8.8mg/kg dsRNA, 8.9mg/kg dsRNA, 9.0mg/kg dsRNA, 9.1mg/kg dsRNA, 9.2mg/kg dsRNA, 9.3mg/kg dsRNA, 9.4mg/kg dsRNA, 9.5mg/kg dsRNA, 9.6mg/kg dsRNA, 9.7mg/kg dsRNA, 9.8mg/kg dsRNA , 9.9mg/kg dsRNA, 9.0mg/kg dsRNA, 10mg/kg dsRNA, 15mg/kg dsRNA, 20mg/kg dsRNA, 25mg/kg dsRNA, 30mg/kg dsRNA, 35mg/kg dsRNA, 40mg/kg dsRNA, A therapeutic amount of iRNA can be administered, such as 45 mg/kg dsRNA, or about 50 mg/kg dsRNA. Values and ranges intermediate to those recited are also intended to be part of the invention.

In certain embodiments, for example, when a composition of the invention comprises a dsRNA and a lipid described herein, the subject receives from about 0.01 mg/kg to about 5 mg/kg, about 0.01 mg/kg. kg to about 10 mg/kg, about 0.05 mg/kg to about 5 mg/kg, about 0.05 mg/kg to about 10 mg/kg, about 0.1 mg/kg to about 5 mg/kg, about 0.1 mg/kg to About 10 mg/kg, about 0.2 mg/kg to about 5 mg/kg, about 0.2 mg/kg to about 10 mg/kg, about 0.3 mg/kg to about 5 mg/kg, about 0.3 mg/kg to about 10 mg /kg, about 0.4 mg/kg to about 5 mg/kg, about 0.4 mg/kg to about 10 mg/kg, about 0.5 mg/kg to about 5 mg/kg, about 0.5 mg/kg to about 10 mg/kg , about 1 mg/kg to about 5 mg/kg, about 1 mg/kg to about 10 mg/kg, about 1.5 mg/kg to about 5 mg/kg, about 1.5 mg/kg to about 10 mg/kg, about 2 mg/kg to About 2.5 mg/kg, about 2 mg/kg to about 10 mg/kg, about 3 mg/kg to about 5 mg/kg, about 3 mg/kg to about 10 mg/kg, about 3.5 mg/kg to about 5 mg/kg, about 4 mg/kg to about 5 mg/kg, about 4.5 mg/kg to about 5 mg/kg, about 4 mg/kg to about 10 mg/kg, about 4.5 mg/kg to about 10 mg/kg, about 5 mg/kg to about 10 mg /kg, about 5.5 mg/kg to about 10 mg/kg, about 6 mg/kg to about 10 mg/kg, about 6.5 mg/kg to about 10 mg/kg, about 7 mg/kg to about 10 mg/kg, about 7. 5 mg/kg to about 10 mg/kg, about 8 mg/kg to about 10 mg/kg, about 8.5 mg/kg to about 10 mg/kg, about 9 mg/kg to about 10 mg/kg, or about 9.5 mg/kg to about A therapeutic amount of iRNA such as 10 mg/kg can be administered. Values and ranges intermediate to those recited are also intended to be part of the invention.

For example, dsRNA may be about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1. 2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2. 5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3. 8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9, 9. 1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, or about 10 mg/kg. Values and ranges intermediate to those recited are also intended to be part of the invention.

In other embodiments, for example, when a composition of the invention comprises a dsRNA described herein and N-acetylgalactosamine, the subject receives about 0.1 to about 50 mg/kg, about 0.25 ~about 50mg/kg, about 0.5 to about 50mg/kg, about 0.75 to about 50mg/kg, about 1 to about 50mg/kg, about 1.5 to about 50mg/kg, about 2 to about 50mg/kg kg, about 2.5 to about 50 mg/kg, about 3 to about 50 mg/kg, about 3.5 to about 50 mg/kg, about 4 to about 50 mg/kg, about 4.5 to about 50 mg/kg, about 5 ~about 50mg/kg, about 7.5 to about 50mg/kg, about 10 to about 50mg/kg, about 15 to about 50mg/kg, about 20 to about 50mg/kg, about 20 to about 50mg/kg, about 25 ~about 50mg/kg, about 25 to about 50mg/kg, about 30 to about 50mg/kg, about 35 to about 50mg/kg, about 40 to about 50mg/kg, about 45 to about 50mg/kg, about 0.1 ~about 45mg/kg, about 0.25 to about 45mg/kg, about 0.5 to about 45mg/kg, about 0.75 to about 45mg/kg, about 1 to about 45mg/kg, about 1.5 to about 45 mg/kg, about 2 to about 45 mg/kg, about 2.5 to about 45 mg/kg, about 3 to about 45 mg/kg, about 3.5 to about 45 mg/kg, about 4 to about 45 mg/kg, about 4 .5 to about 45 mg/kg, about 5 to about 45 mg/kg, about 7.5 to about 45 mg/kg, about 10 to about 45 mg/kg, about 15 to about 45 mg/kg, about 20 to about 45 mg/kg, about 20 to about 45 mg/kg, about 25 to about 45 mg/kg, about 25 to about 45 mg/kg, about 30 to about 45 mg/kg, about 35 to about 45 mg/kg, about 40 to about 45 mg/kg, about 0 .1 to about 40 mg/kg, about 0.25 to about 40 mg/kg, about 0.5 to about 40 mg/kg, about 0.75 to about 40 mg/kg, about 1 to about 40 mg/kg, about 1.5 ~about 40mg/kg, about 2 to about 40mg/kg, about 2.5 to about 40mg/kg, about 3 to about 40mg/kg, about 3.5 to about 40mg/kg, about 4 to about 40mg/kg, About 4.5 to about 40 mg/kg, about 5 to about 40 mg/kg, about 7.5 to about 40 mg/kg, about 10 to about 40 mg/kg, about 15 to about 40 mg/kg, about 20 to about 40 mg/kg kg, about 20 to about 40 mg/kg, about 25 to about 40 mg/kg, about 25 to about 40 mg/kg, about 30 to about 40 mg/kg, about 35 to about 40 mg/kg, about 0.1 to about 30 mg/kg kg, about 0.25 to about 30 mg/kg, about 0.5 to about 30 mg/kg, about 0.75 to about 30 mg/kg, about 1 to about 30 mg/kg, about 1.5 to about 30 mg/kg, About 2 to about 30 mg/kg, about 2.5 to about 30 mg/kg, about 3 to about 30 mg/kg, about 3.5 to about 30 mg/kg, about 4 to about 30 mg/kg, about 4.5 to about 30 mg/kg, about 5 to about 30 mg/kg, about 7.5 to about 30 mg/kg, about 10 to about 30 mg/kg, about 15 to about 30 mg/kg, about 20 to about 30 mg/kg, about 20 to about 30 mg/kg, about 25 to about 30 mg/kg, about 0.1 to about 20 mg/kg, about 0.25 to about 20 mg/kg, about 0.5 to about 20 mg/kg, about 0.75 to about 20 mg/kg kg, about 1 to about 20 mg/kg, about 1.5 to about 20 mg/kg, about 2 to about 20 mg/kg, about 2.5 to about 20 mg/kg, about 3 to about 20 mg/kg, about 3.5 ~ about 20 mg/kg, about 4 to about 20 mg/kg, about 4.5 to about 20 mg/kg, about 5 to about 20 mg/kg, about 7.5 to about 20 mg/kg, about 10 to about 20 mg/kg, Alternatively, a therapeutic amount of iRNA can be administered, such as a dose of about 15 to about 20 mg/kg. In one embodiment, when a composition of the invention comprises a dsRNA described herein and N-acetylgalactosamine, a subject can be administered a therapeutic amount of about 10 to about 30 mg/kg of dsRNA. Values and ranges intermediate to those recited are also intended to be part of the invention.

For example, targets include approximately 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1 .2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2 .5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3 .8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1 , 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4 , 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7 , 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9, 9 .1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10, 10.5, 11, 11.5, 12, 12 .5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19, 19.5, 20, 20.5 , 21, 21.5, 22, 22.5, 23, 23.5, 24, 24.5, 25, 25.5, 26, 26.5, 27, 27.5, 28, 28.5, 29 or about 50 mg. A therapeutic amount of iRNA, such as /kg/kg, can be administered. Values and ranges intermediate to those recited are also intended to be part of the invention.

In certain embodiments of the invention, e.g., a double-stranded RNAi agent is modified (e.g., one or more motifs of three identical modifications to three contiguous nucleotides), e.g., at or near the cleavage site of the agent. When containing one such motif, six phosphorothioate linkages, and a ligand, such agent may be present at about 0.01 to about 0.5 mg/kg, about 0.01 to about 0.4 mg/kg, about 0.01 to about 0.5 mg/kg, about 0.01 to about 0.4 mg/kg, about 0.01 to About 0.3 mg/kg, about 0.01 to about 0.2 mg/kg, about 0.01 to about 0.1 mg/kg, about 0.01 mg/kg to about 0.09 mg/kg, about 0.01 mg/kg kg to about 0.08 mg/kg, about 0.01 mg/kg to about 0.07 mg/kg, about 0.01 mg/kg to about 0.06 mg/kg, about 0.01 mg/kg to about 0.05 mg/kg , about 0.02 to about 0.5 mg/kg, about 0.02 to about 0.4 mg/kg, about 0.02 to about 0.3 mg/kg, about 0.02 to about 0.2 mg/kg, about 0.02 to about 0.1 mg/kg, about 0.02 mg/kg to about 0.09 mg/kg, about 0.02 mg/kg to about 0.08 mg/kg, about 0.02 mg/kg to about 0.07 mg /kg, about 0.02 mg/kg to about 0.06 mg/kg, about 0.02 mg/kg to about 0.05 mg/kg, about 0.03 to about 0.5 mg/kg, about 0.03 to about 0 .4 mg/kg, about 0.03 to about 0.3 mg/kg, about 0.03 to about 0.2 mg/kg, about 0.03 to about 0.1 mg/kg, about 0.03 mg/kg to about 0 .09mg/kg, about 0.03mg/kg to about 0.08mg/kg, about 0.03mg/kg to about 0.07mg/kg, about 0.03mg/kg to about 0.06mg/kg, about 0. 03 mg/kg to about 0.05 mg/kg, about 0.04 to about 0.5 mg/kg, about 0.04 to about 0.4 mg/kg, about 0.04 to about 0.3 mg/kg, about 0. 04 to about 0.2 mg/kg, about 0.04 to about 0.1 mg/kg, about 0.04 mg/kg to about 0.09 mg/kg, about 0.04 mg/kg to about 0.08 mg/kg, about 0.04 mg/kg to about 0.07 mg/kg, about 0.04 mg/kg to about 0.06 mg/kg, about 0.05 to about 0.5 mg/kg, about 0.05 to about 0.4 mg/kg , about 0.05 to about 0.3 mg/kg, about 0.05 to about 0.2 mg/kg, about 0.05 to about 0.1 mg/kg, about 0.05 mg/kg to about 0.09 mg/kg , about 0.05 mg/kg to about 0.08 mg/kg, or about 0.05 mg/kg to about 0.07 mg/kg. Values and ranges intermediate to the foregoing recited values are also intended to be part of the invention; for example, the RNAi agent may be administered to a subject at a dose of about 0.015 mg/kg to about 0.45 mg/kg. can be done.

For example, the RNAi agent, e.g., the RNAi agent in the pharmaceutical composition, can be used at about 0.01 mg/kg, 0.0125 mg/kg, 0.015 mg/kg, 0.0175 mg/kg, 0.02 mg/kg, 0.0225 mg/kg. kg, 0.025mg/kg, 0.0275mg/kg, 0.03mg/kg, 0.0325mg/kg, 0.035mg/kg, 0.0375mg/kg, 0.04mg/kg, 0.0425mg/kg, 0.045mg/kg, 0.0475mg/kg, 0.05mg/kg, 0.0525mg/kg, 0.055mg/kg, 0.0575mg/kg, 0.06mg/kg, 0.0625mg/kg, 0. 065mg/kg, 0.0675mg/kg, 0.07mg/kg, 0.0725mg/kg, 0.075mg/kg, 0.0775mg/kg, 0.08mg/kg, 0.0825mg/kg, 0.085mg/ kg, 0.0875mg/kg, 0.09mg/kg, 0.0925mg/kg, 0.095mg/kg, 0.0975mg/kg, 0.1mg/kg, 0.125mg/kg, 0.15mg/kg, 0.175mg/kg, 0.2mg/kg, 0.225mg/kg, 0.25mg/kg, 0.275mg/kg, 0.3mg/kg, 0.325mg/kg, 0.35mg/kg, 0. It may be administered at a dose of 375 mg/kg, 0.4 mg/kg, 0.425 mg/kg, 0.45 mg/kg, 0.475 mg/kg, or about 0.5 mg/kg. Values intermediate to those listed above are also intended to be part of this invention.

In some embodiments, the RNAi agent is about 100 mg to about 900 mg, such as about 100 mg to about 850 mg, about 100 mg to about 800 mg, about 100 mg to about 750 mg, about 100 mg to about 700 mg, about 100 mg to about 650 mg, about 100mg to about 600mg, about 100mg to about 550mg, about 100mg to about 500mg, about 200mg to about 850mg, about 200mg to about 800mg, about 200mg to about 750mg, about 200mg to about 700mg, about 200mg to about 650mg, about 200mg to about about 600 mg, about 200 mg to about 550 mg, about 200 mg to about 500 mg, about 300 mg to about 850 mg, about 300 mg to about 800 mg, about 300 mg to about 750 mg, about 300 mg to about 700 mg, about 300 mg to about 650 mg, about 300 mg to about 600 mg , about 300 mg to about 550 mg, about 300 mg to about 500 mg, about 400 mg to about 850 mg, about 400 mg to about 800 mg, about 400 mg to about 750 mg, about 400 mg to about 700 mg, about 400 mg to about 650 mg, about 400 mg to about 600 mg, about Administered as a fixed dose of 400 mg to about 550 mg, or between about 400 mg to about 500 mg.

In some embodiments, the RNAi agent is about 100 mg, about 125 mg, about 150 mg, about 175 mg, 200 mg, about 225 mg, about 250 mg, about 275 mg, about 300 mg, about 325 mg, about 350 mg, about 375 mg, about 400 mg, about 425mg, about 450mg, about 475mg, about 500mg, about 525mg, about 550mg, about 575mg, about 600mg, about 625mg, about 650mg, about 675mg, about 700mg, about 725mg, about 750mg, about 775mg, about 800mg, about 825mg, Administered as a fixed dose of about 850 mg, about 875 mg, or about 900 mg.

The iRNA is delivered for a predetermined period of time, such as 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or about 25 minutes. may be administered by intravenous infusion over a period of time. Administration can be repeated periodically, such as weekly, biweekly (ie, every two weeks) for a month, two months, three months, four months, or more. After the initial treatment regimen, therapeutic agents may be administered less frequently. For example, after weekly or biweekly administration for three months, administration can be repeated once a month for six months or a year or more.

Administration of iRNA may result in the presence of serum and/or liver HBV ccc DNA, the presence of serum and/or liver HBV antigens, such as HBsAg and/or HBeAg, in the patient's cells, tissues, blood, urine or other compartments, ALT levels. , and/or AST levels of at least about 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%. , 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35 %, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68% , 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85 %, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least about 99% or more, For example, it can be reduced below the detection level of the assay.

Administration of iRNA reduces the presence of serum and/or liver anti-HBV antibodies in, e.g., cells, tissues, blood, urine, or other compartments of a patient by at least about 5%, 6%, 7%, 8%, 9%, 10%. , 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27 %, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60% , 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77 %, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, It can be increased by 94%, 95%, 96%, 97%, 98%, or at least about 99% or more.

Prior to administering the full dose of iRNA, a smaller dose, such as a 5% infusion, may be administered to the patient and monitored for adverse effects such as allergic reactions. In another example, patients may be monitored for undesirable immunostimulatory effects, such as increased levels of cytokines (eg, TNF-α or INF-α).

Due to the inhibitory effect on HBV expression, the composition according to the invention or the pharmaceutical composition prepared therefrom can improve the quality of life.

The iRNA of the invention may be administered in "naked" form, with modified or unmodified iRNA agents suspended directly in an aqueous or suitable buffer medium as "free iRNA." Free iRNA is administered in the absence of a pharmaceutical composition. Free iRNA can be in a suitable buffer. The buffer may contain acetate, citrate, prolamine, carbonate, or phosphate or any combination thereof. In one embodiment, the buffer is phosphate buffered saline (PBS). The pH and osmolarity of the buffer containing the iRNA can be adjusted as appropriate for administration to a subject.

Alternatively, the iRNA of the invention can be administered as a pharmaceutical composition, such as a dsRNA liposome formulation.

Subjects who may benefit from reducing and/or inhibiting HBV gene expression are those with HBV infection and/or HBV-related diseases or disorders as described herein.

Treatment of subjects that may benefit from reduction and/or inhibition of HBV gene expression includes therapeutic treatment and prophylactic treatment.

The present invention further provides that HBV gene expression can be improved in combination with other pharmaceutical agents and/or other treatments, such as those currently used to treat these disorders. Provided are methods and uses of iRNA agents or pharmaceutical compositions thereof to treat subjects who may benefit from the reduction and/or inhibition of HBV, such as subjects with HBV-related diseases.

For example, in certain embodiments, an iRNA targeting one or more HBV genes is administered in combination with an agent useful for treating an HBV-related disease, e.g., as described elsewhere herein. Ru. For example, additional therapeutic agents and methods suitable for treating subjects who may benefit from reduced HBV expression, such as subjects with HBV-related diseases, include iRNA agents that target different parts of the HBV genome; Antivirals, reverse transcriptase inhibitors (e.g., tenofovir disoproxil fumarate (TDF), tenofovir alafenamide, lamivudine, adefovir dipivoxil, entecavir (ETV), telbivudine, and AGX-1009), immunostimulants (e.g., pegylated interferon α2a (PEG-IFN-α2a), interferon α-2b, recombinant human interleukin-7, and Toll-like receptor 7 (TLR7) agonists), therapeutic vaccines (e.g., GS-4774, DV -601, and TG1050), viral entry inhibitors (e.g., Myrcludex), oligonucleotides that inhibit HbsAg secretion or release (e.g., REP 9AC), capsid inhibitors (e.g., Bay41-4109 and NVR-1221), cccDNA Inhibitors (eg, IHVR-25), or other therapeutic agents and/or treatments to treat HBV-related diseases, such as liver transplantation, chemotherapy, combinations of any of the foregoing.

In certain embodiments, a first iRNA agent that targets one or more HBV genes is administered in combination with a second iRNA agent that targets a different portion of the HBV genome. For example, a first iRNA agent that targets one or more structural genes can be administered in conjunction with a second RNAi agent that targets the X gene. For example, the first RNAi agent includes a first sense strand and a first antisense strand forming a double-stranded region, substantially all of the nucleotides of the first sense strand and the first antisense strand. Substantially all of the nucleotides of the strand are modified nucleotides, the first sense strand is conjugated to a ligand attached at the 3' end, and the ligand is a divalent or trivalent branched linker. and the second RNAi agent includes a second sense strand and a second antisense strand forming a double-stranded region; Substantially all of the nucleotides of the strand and substantially all of the nucleotides of the second antisense strand are modified nucleotides, the second sense strand is conjugated at the 3' end to a bound ligand; is one or more GalNAc derivatives linked via a divalent or trivalent branched linker; the first sense strand is
5'-UCGUGGUGGACUUCUCUCA-3' (SEQ ID NO: 5),
5'-GUGCACUUCGCUUCACCUCUA-3' (SEQ ID NO: 7),
5'-CGUGGUGGACUUCUCUCAAUU-3' (SEQ ID NO: 9),
5'-CGUGGUGGCUUCUCUAAAUU-3' (SEQ ID NO: 37),
5'-GGUGGACUUCUCUCAAUUUUA-3' (SEQ ID NO: 11); and 5'-GUGUGCACUUCGCUUCACA-3' (SEQ ID NO: 39); 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical nucleotide sequences), and the first and second antisense strands each hand,
5'-UGAGAGAAGUCCACCACGAUU-3' (SEQ ID NO: 6);
5'-UAGAGGUGAAGCGAAGUGCACUU-3' (SEQ ID NO: 8);
5'-AAUUGAGAGAAGUCCACCAGCAG-3' (SEQ ID NO: 10);
5'-AAUUGAGAGAAGUCCACCAGCUU-3' (SEQ ID NO: 38),
5'-UAAAAUUGAGAGAAGUCCACCAC-3' (SEQ ID NO: 12), and 5'-UGUGAAGCGAAGUGCACACUU-3' (SEQ ID NO: 40); 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical nucleotide sequences), thereby treating a subject.

In one embodiment, all of the nucleotides of the first and second sense strands and/or all of the nucleotides of the first and second antisense strands include the modification.

In one embodiment, at least one of the modified nucleotides is a 3' terminal deoxythymine (dT) nucleotide, a 2'-O-methyl modified nucleotide, a 2'-fluoro modified nucleotide, a 2'-deoxy modified nucleotide, a fixed nucleotide, a Fixed nucleotides, conformationally restricted nucleotides, constrained ethyl nucleotides, non-basic nucleotides, 2'-amino modified nucleotides, 2'-O-allyl modified nucleotides, 2'-C-alkyl modified nucleotides, 2'- Hydroxly modified nucleotides, 2'-methoxyethyl modified nucleotides, 2'-O-alkyl modified nucleotides, morpholino nucleotides, phosphoramidates, nucleotides containing unnatural bases, tetrahydropyran modified nucleotides, 1,5-anhydro selected from the group consisting of hexitol modified nucleotides, cyclohexenyl modified nucleotides, nucleotides containing phosphorothioate groups, nucleotides containing methylphosphonate groups, nucleotides containing 5'-phosphate, and nucleotides containing 5'-phosphate mimetics.

In certain embodiments, a first iRNA agent that targets one or more HBV genes is administered in combination with a second iRNA agent that targets a gene different from the one or more HBV genes. For example, an iRNA agent that targets one or more HBV genes can be administered in combination with an iRNA agent that targets the CD274/PD-L1 gene. Examples of iRNA agents that target the CD274/PD-L1 gene are described in WO 2011/127180, the entire contents of which are incorporated herein by reference. A first iRNA agent that targets one or more HBV genes and a second iRNA agent that targets a gene different from one or more HBV genes, such as the CD274/PD-L1 gene and/or the HDV gene. , may be administered as part of the same pharmaceutical composition. Alternatively, a first iRNA agent that targets one or more HBV genes and a second iRNA agent that targets a gene different from the one or more HBV genes, such as the CD274/PD-L1 gene and/or the HDV gene. may be administered as part of a different pharmaceutical composition.

CD274 or PD-L1 is a 290 amino acid type I transmembrane protein encoded by the CD274 gene on mouse chromosome 19 and human chromosome 9. CD274/PD-L1 expression is associated with, for example, viruses (e.g., including HIV, HBV, HCV and HTLV, among others), bacteria (e.g., including, inter alia, Helicobacter pylori) and parasites (e.g., Schistosoma mansoni). mansoni) is involved in the evasion of immune responses associated with chronic infections.

PD-L1 associates with PD-1 or B7-1 (CD80) and can influence the immune response by modifying TCR or BCR signaling, but also signals PD-L1-expressing cells. That is, reverse signal transduction via PD-L1 is also possible. Surface plasmon resonance studies have shown a specific and unique interaction between both PD-L1 and B7-1 with an affinity of 1.7 μM, and an interaction between PD-L1 and PD-1. The effect is an affinity of 0.5 μM. Chemical cross-linking studies indicate that PD-L1 and B7-1, like PD-L1 and PD-1, can also interact through their IgV-like domains. The PD-L1:B7-1 interface at least partially overlaps with the putative PD-L1:PD-1 interface. B7-1:PD-L1 interaction can induce inhibitory signals to T cells. Ligating PD-L1 to CD4 T cells by B7-1 or B7-1 to CD4 T cells by PD-L1 sends a functionally significant inhibitory signal. Since both PD-L1 and B7-1 are expressed on T cells, B cells, DCs, and macrophages, there is a bidirectional interaction between B7-1 and PD-L1 on these cell types. There is a possibility that there is. In addition, PD-L1 on non-hematopoietic cells can interact with B7-1 as well as PD-1 on T cells to regulate cells (Keir ME et al., 2008. Annu Rev Immunol. 26:677- 704).

In chronic human viral infections, HIV-specific (Petrovas C et al., 2006, J. Exp. Med. 203:2281-92; Day CL et al., 2006, Nature 443:350-54; Trautmann L et al. ., 2006, Nat. Med. 12:1198-202), HBV-specific (Boettler T et al., 2006, J. Virol. 80: 3532-40; Boni C et al. 2007, J. Virol. 81: Several groups have shown that PD-1 expression is high in HCV-specific T cells (Urbani S et al., 2006, J. Virol. 80:11398-403). PD-L1 also affects peripheral blood CD14+ monocytes and bone marrow DCs of chronic HBV-infected patients (Chen L et al., 2007, J. Immunol. 178:6634-41; Ceng L et al., 2006, J. Viral Hepatitis). .13:725-33), and also in CD14+ cells and T cells of HIV patients (Trabattoni D et al., 2003. Blood 101:2514-20). Blocking the PD-LPD-L interaction in vitro results in HIV-specific, HBV-specific (Boni C et al. 2007, J. Virol. 81:4215-25), HCV-specific, and SIV-specific (Velu V et al., 2007, J. Virol. 81:5819-28) CD8 and CD4 T cell depletion is reversed and proliferation and cytokine production are restored (Petrovas C et al., 2006, J. Exp. Med. 203:2281-92; Day CL et al., 2006, Nature 443:350-54; Trautmann L et al., 2006, Nat. Med. 12:1198-202; Urbani S et al., 2006, J. Virol .80:11398-403). Recent studies have shown that the nucleocapsid protein HCV core can upregulate PD-1 and PD-L1 expression on healthy donor T cells and that upregulation of PD-1 is associated with HCV core and the complement receptor C1QBP. (Yao ZQ et al., 2007, Viral Immunol. 20:276-87).

The subject receiving the first RNAi agent or the first and second RNAi agents of the invention may also be administered one or more other therapeutic agents that function by a non-iRNA mechanism and are useful for treating HBV infection. can be done. Exemplary therapeutic agents that may be used in the combination therapy of the invention include, for example, stimulating the immune system by enhancing T cell helper activity, maturing B lymphocytes, inhibiting T cell suppressors, and enhancing HLA type I expression. Examples include immunomodulatory drugs that Suitable immunomodulators include interferons, which have a variety of properties including antiviral, immunomodulatory, and antiproliferative effects.

For example, the current treatment for chronic hepatitis B is interferon therapy, which is based on at least 6 months of confirmed HBV infection, elevated liver enzymes (AST and ALT), and the presence of virus that is actively dividing in the blood. (HBeAg and/or HBV DNA positive result). Interferon-alpha therapy results in prolonged and persistent disease in approximately 35% of patients with chronic hepatitis B, with normalization of liver enzymes and disappearance of three markers of active infection (HBeAg, HBV DNA, and HBsAg). produce a significant remission. Subjects with acute HBV infection, end-stage cirrhosis, or other serious medical problems are typically not treated with interferon.

In addition, interferon therapy for patients with HBV-related cirrhosis significantly reduces hepatocellular carcinoma (HCC) rates, especially in patients with high amounts of serum HBV DNA. In patients with HBeAg-positive compensated cirrhosis, virological and biochemical remission after interferon therapy is associated with improved survival. In patients with chronic HBV infection, clearance of HBeAg after treatment with interferon-α is associated with improved clinical outcome.

The standard treatment period is considered to be 16 weeks. Patients who exhibit low levels of viral replication at the end of standard regimens benefit most from long-term treatment.

Other exemplary therapeutic agents that may be used in the combination therapy of the invention include, for example, antivirals, nucleotide analogs, nucleoside analogs, reverse transcriptase inhibitors (e.g., tenofovir disoproxil fumarate (TDF ), tenofovir alafenamide, lamivudine, adefovir dipivoxil, entecavir (ETV), telbivudine, AGX-1009, emtricitabine, clevudine, ritonavir, dipivoxil, lobcavir, famvir, FTC, N-acetyl-cysteine (NAC), PC1323, Teradime - HBV (theradigm-HBV), thymosin-α, and ganciclovir), immunostimulants (e.g. pegylated interferon α2a (PEG-IFN-α2a), interferon α-2b, recombinant human interleukin-7, and Toll-like receptor 7 (TLR7) agonists), therapeutic vaccines (e.g., GS-4774, DV-601, and TG1050), viral entry inhibitors (e.g., Myrcludex), oligonucleotides that inhibit HbsAg secretion or release (e.g., REP 9AC), capsid inhibitors (e.g., Bay41-4109 and NVR-1221), cccDNA inhibitors (e.g., IHVR-25), or other therapeutic agents and/or treatments to treat HBV-related diseases, e.g., liver transplantation. , chemotherapy, and combinations of any of the foregoing.

In one embodiment, the method of the invention comprises administering a reverse transcriptase inhibitor to a subject having an HBV infection and/or an HBV-related disease. In another embodiment, the method of the invention comprises administering a reverse transcriptase inhibitor and an immunostimulant to a subject having an HBV infection and/or an HBV-related disease.

The one or more iRNA agents and the additional therapeutic agent and/or treatment may be administered at the same time and/or in the same combination, e.g., parenterally, or the additional therapeutic agent may be administered as part of separate compositions. It can be administered as a single portion or at separate times and/or by other methods known in the art or described herein.

The invention also provides methods of reducing and/or inhibiting HBV expression in cells using iRNA agents of the invention and/or compositions comprising iRNA agents of the invention. In another aspect, the invention provides an iRNA of the invention and/or a composition comprising an iRNA of the invention for use in reducing and/or inhibiting HBV gene expression in a cell. In yet another aspect, there is provided the use of an iRNA of the invention and/or a composition comprising an iRNA of the invention for the manufacture of a medicament that reduces and/or inhibits HBV gene expression in a cell. In yet another aspect, the invention provides an iRNA of the invention and/or a composition comprising an iRNA of the invention for use in reducing and/or inhibiting HBV replication in a cell. In yet another aspect, there is provided the use of an iRNA of the invention and/or a composition comprising an iRNA of the invention for the manufacture of a medicament that reduces and/or inhibits HBV replication in a cell. The present methods and uses include the steps of contacting a cell with an iRNA, such as a dsRNA, of the invention and maintaining the cell for a sufficient period of time to achieve degradation of the HBV gene mRNA transcript, thereby reducing the HBV gene in the cell. or inhibiting HBV replication.

Reduced expression of a gene can be assessed by any method known in the art. For example, reduction in HBV expression can be determined by determining HBV mRNA expression levels using methods well known to those skilled in the art, such as Northern blotting, qRT-PCR, Western blotting, immunological methods, flow cytometry, etc. It may be determined by determining the protein level of HBV using methods well known to those skilled in the art, such as METMETRY, ELISA, etc., and/or by determining the biological activity of HBV.

In the methods and uses of the invention, the cells may be contacted in vitro or in vivo, ie, the cells may be in the subject.

Cells suitable for treatment using the methods of the invention may be any cells that express HBV genes, such as cells infected with HBV or cells that contain an expression vector containing the HBV genome or a portion of an HBV gene. Cells suitable for use in the methods and uses of the invention include mammalian cells, such as primate cells (human cells or non-human primate cells, such as monkey cells or chimpanzee cells), non-primate cells (such as cow cells or chimpanzee cells), cells, pig cells, camel cells, llama cells, horse cells, goat cells, rabbit cells, sheep cells, hamster, guinea pig cells, cat cells, dog cells, rat cells, mouse cells, lion cells, tiger cells, bear cells, or buffalo cells), avian cells (eg duck or goose cells), or whale cells. In one embodiment, the cells are human cells, such as human hepatocytes.

HBV gene expression is at least about 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18% in the cells. , 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35 %, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68% , 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85 %, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or about 100%, i.e. may be inhibited below the detection level of the assay.

HBV replication is at least about 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35% , 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52 %, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85% , 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or about 100%, i.e., assay can be inhibited to below detection levels.

In vivo methods and uses of the invention may include administering to a subject a composition containing an iRNA, wherein the iRNA is complementary to at least a portion of an RNA transcript of an HBV gene in the mammal being treated. nucleotide sequence. When the organism being treated is a human, the composition can be administered, but is not limited to, subcutaneously, intravenously, orally, intraperitoneally, or intracranially (e.g., intraventricularly, intraparenchymally and intrathecally), intramuscularly, Administration may be by any means known in the art, including transdermal, respiratory (aerosol), nasal, rectal, and parenteral routes, including topical (including buccal and sublingual) administration. In certain embodiments, the composition is administered by subcutaneous or intravenous infusion or injection. In some embodiments, the composition is administered by intravenous infusion or injection. In other embodiments, the composition is administered by intramuscular injection.

In certain embodiments, administration is by depot injection. Depot injections can consistently release iRNA over an extended period of time. Thus, depot injections may reduce the frequency of administration required to obtain a desired effect, eg, a desired inhibition of HBV, or a therapeutic or prophylactic effect. Depot injections can also provide more consistent serum concentrations. Depot injections may include subcutaneous or intramuscular injections. In a preferred embodiment, the depot injection is a subcutaneous injection.

In certain embodiments, administration is by pump. The pump may be an external pump or a surgically implanted pump. In certain embodiments, the pump is a subcutaneously implanted osmotic pump. In other embodiments, the pump is an infusion pump. Infusion pumps may be used for intravenous, subcutaneous, intraarterial, or epidural infusions. In a preferred embodiment, the infusion pump is a subcutaneous infusion pump. In other embodiments, the pump is a surgically implanted pump that delivers the iRNA to the liver.

The method of administration can be selected based on whether local or systemic treatment is desired and on the area to be treated. Routes and sites of administration can be selected to improve targeting.

In one aspect, the invention also provides a method for inhibiting HBV gene expression in a mammal, such as a human. The invention also provides compositions comprising an iRNA, eg, a dsRNA, that targets an HBV gene in a mammalian cell for use in inhibiting the expression of an HBV gene in a mammal. In another aspect, the invention provides the use of an iRNA, such as a dsRNA, that targets an HBV gene in a mammalian cell in the manufacture of a medicament for inhibiting the expression of an HBV gene in a mammal.

The methods and uses include administering to a mammal, e.g., a human, a composition comprising an iRNA, e.g., dsRNA, that targets an HBV gene in cells of the mammal; and degrading the mRNA transcript of the HBV gene. maintaining the mammal for a sufficient period of time to obtain an HBV gene, thereby inhibiting expression of the HBV gene in the mammal.

Reduced expression of the gene can be assessed in peripheral blood samples in subjects administered the iRNA by any method known in the art, such as qRT-PCR as described herein. Reduction in protein production can be assessed by any method known in the art, such as ELISA or Western blotting, as described herein. In one embodiment, a liver needle biopsy sample serves as tissue material for monitoring decreased expression of HBV genes and/or proteins. In another embodiment, a blood sample serves as tissue material for monitoring decreased expression of HBV genes and/or proteins.

In one embodiment, confirmation of RISC cleavage of the target in vivo after administration of the iRNA agent is performed by 5'-RACE or a modification of protocols known in the art (Lasham A et al., (2010) Nucleic Acid Res., 38(3) p-e19) (Zimmermann et al. (2006) Nature 441:111-4).

The invention is further illustrated by the following examples, which are not to be construed as limiting. The entire contents of all references, patents and published patent applications, as well as figures and sequence listings, cited throughout this application are hereby incorporated by reference.

Example 1. Sources of iRNA Synthesis Reagents If a reagent source is not specifically indicated herein, such reagents may be obtained from any molecular biology reagent supplier to quality/purity standards for molecular biology applications. can.

Transcript siRNA Design The choice of siRNA design to target HBV is based on two main factors: a) efficacy, and b) coverage of the large number of published HBV sequences of all known serotypes (A-H). The motivation was the desire to use siRNAs with near perfect match rates of >90%. Coordinates for siRNA selection were determined against the NCBI HBV reference genomic sequence NC_003977.1 (GenBank accession number GI:21326584 (SEQ ID NO: 1). Two flanking regions of the HBV genome encoding surface antigen (HbSAg) and HBV polymerase. A first set of siRNAs containing structure-activity modifications, including various 2'-O-methyl and 2'-fluoro substitution patterns, centered on HbSAg and polymerase were designed, synthesized, and screened in vitro. In addition, a second set of siRNAs targeting additional target regions was designed, synthesized, and screened, with particular attention to positions 1581-1599 of SEQ ID NO: 1, which encodes the X gene.

A detailed list of unmodified HBV sense and antisense strand sequences is shown in Table 3.

A detailed list of modified HBV sense and antisense strand sequences is shown in Table 4.

siRNA Synthesis 1 μmol scale HBV siRNA sequences were synthesized on a Mermade 192 synthesizer (BioAutomation) using solid support-mediated phosphoramidite chemistry. The solid support was a controlled pore glass (500A) loaded with a custom GalNAc ligand or a universal solid support (AM biochemical). Auxiliary synthetic reagents, 2'-F and 2'-O-methyl RNA and deoxyphosphoramidites were obtained from Thermo-Fisher (Milwaukee, WI) and Hongene (China). 2'F2'-O-methyl, GNA (glycol nucleic acid), 5' phosphate and abasic modifications were introduced using the corresponding phosphoramidites. Synthesis of 3'GalNAc conjugate single strands was performed on a GalNAc-modified CPG support. A custom CPG universal solid support was used for the synthesis of antisense single strands. The coupling time for all phosphoramidites (100 mM in acetonitrile) was 5 minutes, and 5-ethylthio-1H-tetrazole (ETT) was used as the activator (0.6 M in acetonitrile). 3-((dimethylamino-methylidene)amino)-3H-1,2,4-dithiazole-3-thione (DDTT, from Chemgenes (Wilmington, MA, USA) in anhydrous acetonitrile/pyridine (1:1 v/v) A phosphorothioate bond was created using a 50mM solution of (obtained). The oxidation time was 3 minutes. All sequences were finally synthesized with the DMT group removed ("DMT off").

After completion of solid phase synthesis, oligoribonucleotides were cleaved from the solid support and deprotected using 200 μL aqueous methylamine reagent at 60° C. for 20 minutes in a sealed 96 deep-well plate. At the end of the cleavage and deprotection steps, the synthesis plate was allowed to reach room temperature and precipitated by adding 1 mL of acetontile:ethanol mixture (9:1). The plate was cooled at −80° C. for 2 hours and the supernatant was carefully decanted using a multichannel pipette. The oligonucleotide pellet was resuspended in 20 mM NaOAc buffer and desalted using a 5 mL HiTrap size exclusion column (GE Healthcare) on an AKTA Purifier System equipped with an A905 autosampler and Frac 950 fraction collector. Desalted samples were collected into 96-well plates. Samples from each array were analyzed by LC-MS to confirm identity, quantified by UV (260 nm), and purity of selected sample sets determined by IEX chromatography.

Annealing of HBV single strands was performed on a Tecan liquid handling robot. Equimolar mixtures of sense and antisense single strands were combined and annealed in a 96-well plate. After combining the complementary single strands, the 96-well plate was tightly sealed and heated in an oven at 100° C. for 10 minutes, allowing it to slowly reach room temperature over a period of 2-3 hours. The concentration of each duplex was standardized to 10 μM in 1×PBS.

Example 2. In vitro screening of siRNA duplexes Cell culture and transfection Cos7 cells (ATCC, Manassas, VA) were grown to near confluence at 37°C in an atmosphere of 5% CO in DMEM (ATCC) supplemented with 10% FBS. Afterwards, it was removed from the plate by trypsinization. A Dual-Glo® luciferase construct made in a psiCHECK2 plasmid containing approximately 1.1 kb of HBV genomic sequence was grown into approximately 15 x 104 cells using Lipofectamine 2000 (Invitrogen, Carlsbad CA. Cat. No. 11668-019). transfected. For each well of a 96-well plate, 0.2 μl of Lipofectamine was added to 10 ng of plasmid vector in 14.8 μl of Opti-MEM and allowed to complex for 15 minutes at room temperature. This mixture was then added to the cells resuspended in 80 μl of fresh complete medium. Approximately 24 hours later, the medium was removed and cells were re-transfected with siRNA. Each siRNA was transfected into cells that had already been transfected with the psiCHECK2-HBV vector with a perfect match for the siRNA. For siRNA transfection, add 14.8 μl of Opti-MEM + 0.2 μl of Lipofectamine RNAiMax (Invitrogen, Carlsbad CA. Cat. No. 13778-150) per well to 5 μl of siRNA duplex per well in a 96-well plate. and incubated for 15 minutes at room temperature. This mixture was then added to cells that had already been transfected with a psiCHECK2-HBV plasmid with a perfect match to the siRNA sequence. After incubating cells for 24 hours, luciferase was measured.

Single dose experiments were performed at 10 nM and 0.01 nM final duplex concentrations.

Dual-Glo® Luciferase Assay Firefly luciferase (transfection control) and Renilla luciferase (fused to the HBV target sequence) were measured 24 hours after siRNA transfection. First, the medium was removed from the cells. Firefly luciferase activity was then measured by adding 75 μl of Dual-Glo® luciferase reagent equal to the culture medium volume to each well and mixing. After incubating the mixture at room temperature for 30 minutes, firefly luciferase signals were detected by measuring luminescence (500 nm) with Spectramax (Molecular Devices). Renilla luciferase activity was measured by adding 75 μl of room temperature Dual-Glo® Stop & Glo® reagent to each well, incubating the plate for 10-15 minutes, and then measuring the luminescence again. Shiitake luciferase signal was determined. The Dual-Glo® Stop & Glo® reagent quenches the firefly luciferase signal and maintains the luminescence of the Renilla luciferase reaction. siRNA activity was determined by normalizing the Renilla (HBV) signal to the firefly (control) signal within each well. The magnitude of siRNA activity was then assessed compared to cells transfected with the same vector but not treated with siRNA or treated with non-targeting siRNA. All transfections were performed with n=2 or more.

Table 5 shows the results of a single dose screen in Cos7 cells transfected with the indicated HBV iRNAs. Data are expressed as percent remaining mRNA relative to negative control.

Example 3. Additional siRNA duplex synthesis and in vitro screening Additional iRNA molecules targeting the HBV genome were synthesized as described above. A detailed list of additional unmodified HBV sense and antisense strand sequences is shown in Table 6, and a detailed list of modified HBV sense and antisense strand sequences is shown in Table 7.

Transfect the duplex into HepG2.215 and Hep3B cells to detect primer/probe pairs (PORF-1_A and PORF-1_B) that detect HBV P open reading frame (ORF) RNA and/or HBV S ORF RNA. These double-stranded single-dose screens were performed in duplicate by measuring HBV viral RNA using the primer set (SORF-2_A and SORF-2_B). The results of the HepG2.2.15 cell assay are shown in Table 8 and the results of the Hep3B cell assay are provided in Table 9.

Some of these duplexes were also assayed for in vitro metabolic stability using two assays, a tritosome stability assay and a cytosolic stability assay.

For the tritosome stability assay, rat liver tritosomes (Xenotech custom product PR14044) were thawed to room temperature and diluted to 0.5 units/mL acid phosphatase in 20 mM sodium citrate pH 5.0 buffer. 24-hour samples were prepared by mixing 100 μL of 0.5 units/mL acid phosphatase tritosomes with 25 μL of 0.4 mg/mL siRNA sample in a microcentrifuge tube and incubating for 24 hours in an eppendorf Thermomixer set at 37°C and 300 rpm. was prepared. After 24 hours of incubation, 300 μL of Phenomenex Lysis Loading Buffer (Cat. No. ALO-8498) and 12.5 μL of 0.4 mg/mL internal standard siRNA were added to each sample. By mixing 100 μL of 0.5 units/mL acid phosphatase tritosomes with 25 μL of 0.4 mg/mL siRNA sample, 300 μL of Phenomenex lysis loading buffer, and 12.5 μL of 0.4 mg/mL internal standard siRNA. Samples were prepared at time 0. siRNA was extracted from the 24-hour and 0-hour samples using the Phenomenex Clarity OTX Starter kit (Cat. No. KSO-8494). After extraction of the samples, they were transferred to microcentrifuge tubes and dried using a Labconco CentriVap concentrator (Cat. No. 7810010). The samples were then resuspended in 500 μL of nuclease-free water. 50 μL of each sample was run on an Agilent Technologies 1260 Infinity Binary LC with an Agilent Technologies 6130 quadrupole LC/MS. The quaternary pump method was run at 0.400 mL/min for 12.20 minutes with the following timetable:

The binary pump method was run at 0.700 mL/min for 12.20 minutes with the following timetable:

Both left and right columns were set at 75.00°C. UV signals were measured at 260 nm wavelength. The percentage remaining of each strand was calculated using the following formula:
% remaining chains = 100 x (peak area chains 24 hours/peak area chains 0 hours x (peak area chains 24 hours/peak area chains 0 hours)).

For cytosolic stability assays, female rat liver cytosol (Xenotech catalog number R1500.C) was thawed to room temperature and diluted to 1 mg/mL in 50 mM Tris buffer:HCl pH 7.4, 5 mM MgCl2. A 24-hour sample was prepared by mixing 100 uL of 1 mg/mL cytosol with 25 uL of 0.4 mg/mL siRNA sample in a microcentrifuge tube and incubating for 24 hours in an eppendorf Thermomixer set at 37° C. and 300 rpm. After 24 hours of incubation, 300 uL of Phenomenex Lysis Loading Buffer (Catalog Number ALO-8498) and 12.5 uL of 0.4 mg/mL internal standard siRNA were added to each sample. Prepare the 0-hour sample by mixing 100 uL of 1 mg/mL cytosol with 25 uL of 0.4 mg/mL siRNA sample, 300 uL of Phenomenex lysis loading buffer, and 12.5 uL of 0.4 mg/mL internal standard siRNA. did. siRNA was extracted from the 24-hour and 0-hour samples using the Phenomenex Clarity OTX Starter kit (Cat. No. KSO-8494). After extraction of the samples, they were transferred to microcentrifuge tubes and dried using a Labconco CentriVap concentrator (Cat. No. 7810010). The sample was then resuspended in 500 uL of nuclease-free water. 50 uL of each sample was run on an Agilent Technologies 1260 Infinity Binary LC with an Agilent Technologies 6130 quadrupole LC/MS. The quaternary pump method was run at 0.400 mL/min for 12.20 minutes with the following timetable:

The binary pump method was run at 0.700 mL/min for 12.20 minutes with the following timetable:

Both left and right columns were set at 75.00°C. UV signals were measured at 260 nm wavelength. The percentage remaining of each strand was calculated using the following formula:
% remaining chains = 100 x (peak area chains 24 hours/peak area chains 0 hours x (peak area chains 24 hours/peak area chains 0 hours)).

The results of the 24 hour tritosome stability assay are provided in Table 10 and the results of the 24 hour cytosolic stability assay are provided in Table 11.

Example 4. Synthesis and Screening of Additional siRNA Duplexes Additional iRNA molecules targeting the HBV genome were designed and synthesized as described above. A detailed list of additional unmodified HBV sense and antisense strand sequences is shown in Table 12, and a detailed list of modified HBV sense and antisense strand sequences is shown in Table 13.

A primary single dose screen of these iRNA duplexes was performed using the Dual-Glo® luciferase assay as described above. The results of this screen in Cos7 cells transfected with the indicated HBV iRNAs are shown in Table 14. Data are expressed as percent mRNA remaining relative to negative control at 24 hours.

These duplexes were also assayed for dose response for viral RNA silencing using the Dual-Glo® luciferase assay as described above. The duplex doses used in these assays were: 50 nM, 8.333333333 nM, 1.388888889 nM, 0.231481481 nM, 0.038580247 nM, 0.006430041 nM, 0.001071674 nM, 0.000178612 nM, 2.97687× 10-5nM , 4.96145 x 10-6 nM, 8.26909 x 10-7 nM, and 1.37818E x 10-7 nM (these correspond to 1:6 dilutions of the duplex over 12 doses starting at 50 nM). . The results of this screen in Cos7 cells transfected with the indicated HBV iRNAs are shown in Table 15. Data are expressed as percent mRNA remaining relative to negative control at 24 hours.

The in vitro efficacy and potency of these duplexes was also assayed. In particular, the duplex dose response for silencing of viral RNA in transfected HepG2.2.15 and Hep3B cell lysates and silencing of HBsAg in HepG2.2.15 cell supernatants was determined. Cells were transfected with 12 separate doses of duplex ranging from 50 nM to 1 x 10-7 nM and primer/probe pairs detecting P ORF and/or S ORF were added at 72 hours post-transfection. was used to determine the levels of viral RNA. Levels of HBsAg were determined using an ELISA assay.

Results of PORF viral RNA silencing in HepG2.2.15 cells using the indicated duplexes are provided in Table 16. Results of SORF viral RNA silencing in HepG2.2.15 cells using the indicated duplexes are provided in Table 17. Results of HBsAg silencing in HepG2.2.15 cells are provided in Table 18.

Results of PORF viral RNA silencing in Hep3B cells using the indicated duplexes are provided in Table 19.

These duplexes were also assayed for in vitro stability using two assays, a tritosome stability assay and a cytosolic stability assay, as described above. The results of these assays are provided in Table 20.

Dose-response screens of various combinations of these duplexes were also performed in HepG2.215 cells. The duplex doses used in these assays were: 50 nM, 8.333333333 nM, 1.388888889 nM, 0.231481481 nM, 0.038580247 nM, 0.006430041 nM, 0.001071674 nM, 0.000178612 nM, 2.97687× 10-5nM , 4.96145 x 10-6 nM, 8.26909 x 10-7 nM, and 1.37818E x 10-7 nM (these correspond to 1:6 dilutions of the duplex over 12 doses starting at 50 nM). . At 72 hours after transfection of these duplexes, the levels of viral RNA (P ORF and S ORF) and secreted HBsAg were determined as described above. The results of these assays are provided in Table 21.

Example 5. Synthesis of additional siRNA duplexes and in vitro screening Additional iRNA molecules targeting the X ORF of the HBV genome were designed and synthesized as described above. A detailed list of additional unmodified HBV sense and antisense strand sequences is shown in Table 22. A detailed list of additional modified HBV sense and antisense strand sequences is shown in Table 23.

A single dose screen of these duplexes in 1 nm and 50 nm Cos7 cells was performed using the Dual-Glo® luciferase assay described above. The results of this assay are provided in Table 24.

Based on these assays, RNAi agents targeting five sites of the HBV was designed and synthesized. These additional agents were evaluated in in vitro assays as described above. A detailed list of additional unmodified sense and antisense strand sequences targeting the HBV X ORF is shown in Table 25. A detailed list of additional modified sense and antisense strand sequences targeting the HBV X ORF is provided in Table 26.

These iRNA agents were also evaluated for in vivo efficacy using the AAV-HBV mouse model (see, eg, Yang, et al. (2014) Cell and Mol Immunol 11:71). This mouse model exhibits persistent HBV viremia upon infection with a recombinant adeno-associated virus (AAV) harboring a replication-competent HBV genome. Hepatic expression of HBV genes in these mice mimics HBV infection in humans, and these mice exhibit severe hepatitis and liver damage seen with elevated ALT levels, fibrosis and steatosis.

These AAV-HBV mice received a single 3 mg/kg dose of AD-66808, AD-66809, AD-66810, AD-66811, AD-66812, AD-66813, AD-66814, AD-66815, AD-66816, and AD-66817 were administered subcutaneously, and the HBsAg levels in the serum of the animals were determined before administration and on days 14/15 after administration. The results of these experiments are provided in Figure 2 and Table 27 and demonstrate that serum levels of HBsAg are reduced after a single administration of these agents. Table 27 also provides the results of a single dose screen for the same RNAi agents using the Dual-Glo® luciferase assay as described above in Cos7 cells transfected with the indicated HBV iRNA. Data are expressed as percent mRNA remaining relative to negative control at 24 hours.

Example 6. In Vivo Screening of siRNA Duplexes Some lead iRNA agents were evaluated for in vivo efficacy using the AAV-HBV mouse model described above. AAV-HBV mice were administered a single 3 mg/kg dose of AD-66019, AD-65375, AD-65047, AD-65377, AD-66111, AD-65421, or AD-66110 before and after administration. Serum HBsAg levels of the animals on days 5 and 10 were determined. As a control, AAV-HBV mice were administered a 3 mg/kg dose of dsRNA targeting mouse/rat transthyretin (mrTTR). The results of these experiments are shown in Figure 3 and demonstrate that serum levels of HBsAg are reduced after a single administration of these agents.

Figure 4 is a graph showing the percentage of pre-dose HBsAg remaining on days 5 and 10 in these animals after administration of a single 3 mg/kg dose, which was also determined. The results of these experiments are shown in Figure 4. Figure 4 also shows the percentage of HBsAG remaining at day 10 post-dose versus the percentage of HBsAG remaining at day 10 post-dose in animals administered 3 mg/kg control dsRNA targeting mouse/rat transthyretin (mrTTR). The percentage of remaining HBsAG is also shown.

Based at least in part on the results of the in vitro and in vivo assays described above, AD-65403, which silences three HBV RNAs, and which silences the AD-66810 was selected for shing.

Figure 5 demonstrates that a single 3 mg/kg dose of AD-65403 achieves potent and specific knockdown of HBsAg in the AAV-HBV mouse model of HBV infection. Specifically, a single 3 mg/kg subcutaneous dose of AD-65403 achieves up to a 3.9 log10 reduction in HBsAg levels, with an average reduction in HBsAg of 1.8 log10 5-10 days after a single dose. .

Figures 6A and 6B show that AD-66810 at a single 0.3 mg/kg, 1 mg/kg, 3 mg/kg, or 9 mg/kg subcutaneous dose, especially at higher doses, was tested in the AAV-HBV mouse model of HBV infection. AD-66810 has been demonstrated to achieve potent and specific knockdown of HBsAg. The percent reduction in HBsAg in serum is shown on a standard scale in FIG. 6A and on a log10 scale in FIG. 6B. Figure 7 shows that AD-66810 administered at three weekly subcutaneous 3 mg/kg doses achieves potent and specific knockdown of HBsAg over a period of more than 4 months in an AAV-HBV mouse model of HBV infection. It has been proven that.

Example 7. Treatment of HBV Infection with Combinations of HBV Targeting Agents Some iRNA agents of the invention are evaluated for in vivo efficacy using the AAV-HBV mouse model described above. AAV-HBV mice are administered one or more doses of AD-65403 and AD-66810, either alone or in combination with each other. Exemplary dosing regimens include a single 3 mg/kg total iRNA dose of AD-65403, AD-66810, or a combination of AD-65403 and AD-66810 (i.e., a mixture or a total administered as two separate doses). 1.5 mg/kg of each iRNA agent for 3 mg/kg iRNA); or a single dose of 0.3 mg/kg, 1 mg/kg, 3 mg/kg, or 9 mg/kg total iRNA agent dose of AD-65403, AD -66810, or a combination of AD-65403 and AD-66810. An exemplary multiple dose regimen includes, for example, three weekly administrations of one dose per week using any of the dosage levels provided in the exemplary single dose regimen. As routine in the art, appropriate control iRNA agents are also administered as controls.

HBsAg levels in the animals' serum are determined pre-dose and at pre-determined post-dose intervals, eg every 4 days post-dose, until HBsAg levels return to baseline for all animals. Administration of AD-65403, AD-66810, or a combination of AD-65403 and AD-66810 results in sustained and specific knockdown of serum HBsAg.

Example 8. Treatment of HDV Infection with iRNA Agents Targeting Hepatitis B Virus Hepatitis delta virus (HDV) is a defective RNA virus that requires the help of HBV for its replication and assembly of new virions. Therefore, HDV is infectious only in the presence of active HBV infection. The HDV genome contains only one actively transcribed open reading frame encoding two isoforms of the hepatitis D delta antigen. Post-translational modifications of small and large delta antigens (S-HDAg and L-HDAg), including phosphorylation and isoprenylation, respectively, confer their specific properties to these antigens. Effective HBV treatment also improves HDV infection.

A chimpanzee model of HDV is known. Some iRNA agents of the invention are evaluated for in vivo efficacy using the chimpanzee HDV model or other suitable HDV model. HDV-infected chimpanzees are administered one or more doses of AD-65403 and AD-66810, either alone or in combination with each other. Exemplary dosing regimens include a single 3 mg/kg total iRNA dose of AD-65403, AD-66810, or a combination of AD-65403 and AD-66810 (i.e., administered as a mixture or two separate doses). 1.5 mg/kg of each iRNA agent for a total of 3 mg/kg of iRNA agents); or a single dose of 0.3 mg/kg, 1 mg/kg, 3 mg/kg, or 9 mg/kg total iRNA agent dose of AD-65403 , AD-66810, or a combination of AD-65403 and AD-66810. An exemplary multiple dose regimen includes, for example, three weekly administrations of one dose per week using any of the dosage levels provided in the exemplary single dose regimen. Appropriate control iRNAs are also administered as controls, as is routine in the art.

Determine the levels of one or more of S-HDAg, L-HDAg, and HDV RNA, optionally in conjunction with HBsAg, in the animal's serum before and at pre-dose intervals, e.g. every 4 days, and determine whether the antigen or RNA Monitor levels. Administration of AD-65403, AD-66810, or a combination of AD-65403 and AD-66810 results in sustained and specific knockdown of serum HBsAg, such as S-HDAg, L-HDAg, and HDV RNA. leads to an improvement in HDV as evidenced by one or more statistically significant reductions in . These results demonstrate that administration of one or both of AD-65403 and AD-66810 is effective in treating HDV.

Equivalents Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments and methods described herein. Such equivalents are intended to be covered by the following claims.

Claims (13)

A pharmaceutical composition comprising a double-stranded RNAi agent for use in treating a subject infected with a hepatitis D virus (HDV) infection, wherein the subject has a hepatitis B virus (HBV) infection and an HDV infection. the double- stranded RNAi agent comprises a sense strand and an antisense strand forming a double-stranded region, the sense strand comprising 5'-GUGUGCACUUCGCUUCACA-3' (SEQ ID NO: 39); , and the antisense strand comprises 5'-UGUGAAGCGAAGUGCACACUU-3' (SEQ ID NO: 40),
all of the nucleotides of the sense strand and all of the nucleotides of the antisense strand are modified nucleotides;
the sense strand is conjugated to a ligand attached at the 3' end, and the ligand is one or more GalNAc derivatives attached via a divalent or trivalent branched linker. Pharmaceutical composition. At least one of the modified nucleotides is a deoxynucleotide, a 3' terminal deoxythymine (dT) nucleotide, a 2'-O-methyl modified nucleotide, a 2'-fluoro modified nucleotide, a 2'-deoxy modified nucleotide, a fixed nucleotide, or a non-fixed nucleotide. , conformationally restricted nucleotides, constrained ethyl nucleotides, abasic nucleotides, 2'-amino modified nucleotides, 2'-O-allyl modified nucleotides, 2'-C-alkyl modified nucleotides, 2'-methoxyethyl Modified nucleotides, 2'-O-alkyl modified nucleotides, morpholino nucleotides, phosphoramidates, nucleotides containing unnatural bases, tetrahydropyran modified nucleotides, 1,5-anhydrohexitol modified nucleotides, cyclohexenyl modified nucleotides, phosphorothioates The pharmaceutical composition according to claim 1, which is a nucleotide comprising a methylphosphonate group, a nucleotide comprising a 5'-phosphate group, or a nucleotide comprising a 5'-phosphate mimetic. 3. The pharmaceutical composition of claim 2, wherein the 5'-phosphate mimetic is 5'-vinyl phosphate (5'-VP). The sense strand comprises 5'-gsusguGfcAfCfUfucgcuucaca-3' (SEQ ID NO: 41), and the antisense strand comprises 5'-usGfsugaAfgCfGfaaguGfcAfcacsusu-3' (SEQ ID NO: 42) [wherein a, g, c and u are 2'-O-methyl (2'-OMe) A, 2'-OMeG, 2'-OMeC and 2'-OMeU, respectively; Af, Cf, Gf and Uf are 2'-fluoro A, 2'-fluoro C, 2'-fluoro G and 2'-fluoro U; and s is a phosphorothioate bond], the pharmaceutical composition according to claim 1. The ligand is

The pharmaceutical composition according to any one of claims 1 to 4, which is The RNAi agent is shown in the following schematic diagram.

6. A pharmaceutical composition according to claim 5, conjugated to a ligand of the formula: wherein X is O or S. The sense strand comprises 5'-gsusguGfcAfCfUfucgcuucacaL96-3' (SEQ ID NO: 1275), and the antisense strand comprises 5'-usGfsugaAfgCfGfaaguGfcAfcacsusu-3' (SEQ ID NO: 1285) [wherein a, c, g and u are 2'-O-methyl (2'-OMe) A, 2'-OMeC, 2'-OMeG and 2'-OMeU, respectively; Af, Cf, Gf and Uf are 2'-fluoro A, 2'-fluoro C, 2'-fluoro G and 2'-fluoro U; s is a phosphorothioate bond; and L96 is N-[tris(GalNAc-alkyl)-amidodecanoyl)]-4 -hydroxyprolinol], the pharmaceutical composition according to claim 1. A pharmaceutical composition according to any one of claims 1 to 7 , further comprising a pharmaceutically acceptable carrier . 9. A pharmaceutical composition according to claim 8, wherein the pharmaceutically acceptable carrier is a non-buffered or buffered solution. The double-stranded RNAi agent has a body weight-based dose of about 0.01 mg/kg to about 10 mg/kg or about 0.5 mg/kg to about 50 mg/kg; about 10 mg/kg to about 30 mg/kg based on body weight. The pharmaceutical composition according to any one of claims 1 to 9, administered in a dose; a body weight based dose of about 3 mg/kg; a body weight based dose of about 10 mg/kg; or a constant dose of about 50 mg to 200 mg. thing. The pharmaceutical composition according to any one of claims 1 to 10 , wherein the double-stranded RNAi agent is administered subcutaneously or intravenously. A pharmaceutical composition according to any one of claims 1 to 11 , wherein the subject is administered a further therapeutic agent. The additional therapeutic agent may include an antiviral agent, a reverse transcriptase inhibitor, an immunostimulant, a therapeutic vaccine, a virus entry inhibitor, an oligonucleotide that inhibits the secretion or release of HbsAg, a capsid inhibitor, a covalently closed circular (ccc) 13. A pharmaceutical composition according to claim 12 , which is an HBV DNA inhibitor, or a combination of any of the foregoing.

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